BRAIN,BEHAVIOR,

Brain, Behavior, and Immunity 18 (2004) 349–360

and IMMUNITY

www.elsevier.com/locate/ybrbi

Nocturnal proinflammatory cytokine-associated sleep disturbancesin abstinent African American alcoholics

Michael Irwin,* Gina Rinetti, Laura Redwine, Sarosh Motivala,Jeff Dang, and Cindy Ehlers

Cousins Center for Psychoneuroimmunology, Neuropsychiatric Institute, University of California, Los Angeles, CA, USA

Received 11 December 2003; received in revised form 21 January 2004; accepted 3 February 2004

Available online 5 March 2004

Abstract

Animal studies reveal that cytokines play a key role in the regulation of sleep. Alcoholic patients show profound alterations of

sleep and a defect in the homeostatic recovery of sleep following sleep loss. In this study, we investigated whether nocturnal

plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-a (TNF) were associated with disordered sleep in alcohol de-

pendence by testing the temporal relationships between these inflammatory cytokines and sleep, before and after sleep deprivation.

All-night polysomnography and serial blood sampling at 23:00, 03:00, and 06:30 h were conducted across baseline, partial sleep

deprivation, and recovery nights in abstinent African American alcoholics (n ¼ 16) and matched controls (n ¼ 15). Coupled with

prolonged sleep latency and increased rapid eye movement sleep, alcoholics showed nocturnal elevations of IL-6 and TNF as

compared to controls after adjustment for alcohol consumption and body mass index. Following sleep deprivation, alcoholics

showed greater nocturnal levels of IL-6 and greater nocturnal increases of TNF as compared to controls. Pre-sleep IL-6 levels at

23:00 h correlated with prolonged sleep latency after adjustment for potential confounders whereas IL-6 levels at 03:00 h correlated

with rapid eye movement sleep in the second half of the night. Taken together, these findings indicate that circulating levels of

proinflammatory cytokines may have a negative influence on sleep initiation. These findings have implications for determining why

sleep is disordered in alcoholics and may aid in the development of novel treatments to optimize sleep in this population.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Alcohol; Alcohol dependence; Sleep; Cytokines; Inflammation; Sleep deprivation; African American; Immunity; Body mass index

1. Introduction

Sleep disturbance is one of the most prominent

complaints of alcoholic patients with over 70% of al-

cohol dependent person reporting sleep problems that

fail to resolve over the course of alcoholic recovery

(Brower et al., 1998; Drummond et al., 1998). EEGsleep studies reveal a reduction of total sleep time,

fragmentation of sleep, loss of Stages 3 and 4 (delta)

sleep, and increases of rapid eye movement (REM)

sleep in recovering alcohol dependent patients (Gillin

et al., 1990a,b; Irwin et al., 2000). Moreover, alcoholics

show a defect in homeostatic regulation of sleep; when

sleep regulatory processes are naturalistically probed

* Corresponding author. Fax: 1-310-794-7247.

E-mail address: mirwin1@ucla.edu (M. Irwin).

0889-1591/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbi.2004.02.001

by sleep deprivation, alcoholics are unable to generate

an increase of slow wave sleep as is usually seen fol-

lowing early night partial sleep deprivation (Irwin

et al., 2002). These findings are particularly pronounced

in African American alcoholics; prior studies have

found more severe disturbances of EEG sleep and

greater abnormalities of immune measures as com-pared to European American alcoholics (Irwin and

Miller, 2000; Irwin et al., 2000). Increasing evidence

also implicates disordered sleep as a risk factor for

alcoholic relapse (Clark et al., 1999; Gillin et al., 1994).

Furthermore, recent data show that sleep complaints

and abnormal EEG sleep (e.g., prolonged sleep la-

tency) predict future physical health problems, and are

associated with a nearly 2-fold increase in mortalityrisk in community samples (Dew et al., 2003; Kripke

et al., 2002; Mallon et al., 2002). Despite the high

350 M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360

prevalence and severity of sleep abnormalities in alco-holics, the neurobiological mechanisms that underlie

abnormal sleep in this at-risk population are not

known. In this study, we provide evidence that the

complex cytokine network is one system that might

contribute to declines in sleep continuity and abnormal

sleep architecture in alcoholics.

Basic observations in rodents demonstrate that cy-

tokines have a physiological role in the regulation ofsleep (Krueger and Toth, 1994; Opp and Toth, 2003),

although there has been limited effort to translate these

data into clinical populations with disordered sleep.

Inflammatory cytokines such as interleukin-6 (IL-6) and

tumor necrosis factor-a (TNF) are cellular signaling

peptides that regulate the immune response, play a

central role in host defense, and mediate endocrine and

physiological responses by virtue of their communica-tion with the brain by direct neural paths (Papanicolaou

et al., 1998; Watkins and Maier, 1999). While elevations

of these cytokines are associated with a constellation of

behavioral symptoms such as fatigue, depressed mood,

and decreases in cognitive performance collectively

known as ‘‘sickness behaviors’’ (Dantzer, 2001; Kronfol

and Remick, 2000), less is known about the effects of IL-

6 and TNF on sleep in humans. To our knowledge, nostudies have evaluated whether circulating levels of these

proinflammatory cytokines are related to abnormal

sleep in alcoholics. However, one study found that ab-

stinent alcoholics show elevations in stimulated IL-6

production (Song et al., 1999), and other studies suggest

that sleep deprivation and disordered sleep leads to

daytime increases of this cytokine (Vgontzas et al., 1997,

1999). In contrast, exogenous doses of IL-6 reduce deltasleep in the first half of the night in healthy adults

(Spath-Schwalbe et al., 1998). In rats, acute adminis-

tration of IL-6 produced sleep fragmentation (Hogan

et al., 2003).

In this study, we tested the hypothesis that elevated

circulating levels of the proinflammatory cytokines IL-

6 and TNF are associated with disordered sleep in

alcohol dependence. Early night partial sleep depri-vation (PSD-E) was used as a probe of sleep regula-

tory processes (Irwin et al., 1992, 1996). IL-6 and

TNF were repeatedly sampled across the nocturnal

period to test the reciprocal influences of inflamma-

tory cytokines and sleep during baseline, PSD-E, and

recovery nights. We predicted that alcoholics would

show higher nocturnal levels of circulating IL-6 and

TNF as compared to controls. Second, in alcoholicswho showed disordered sleep and abnormal recovery

of sleep following sleep deprivation (Irwin et al.,

2002), administration of PSD-E would lead to further

nocturnal elevations of these proinflammatory cyto-

kines. Third, circulating levels of IL-6 and TNF would

temporally correlate with measures of sleep continuity

and architecture.

2. Materials and methods

2.1. Human subjects

A total of 50 African American men (25 control

subjects, 25 alcoholic patients) fulfilled screening eligi-

bility criteria, gave informed consent, and entered the

research protocol. Of this screened sample, the following

subjects were excluded: 4 controls due to medication use(e.g., b-blockers, nonsteroidal anti-inflammatory agents,

steroids), 1 control and 7 alcoholics due to positive

substance use screen during the sleep protocol or within

the 2 week period prior to assessment, 5 control subjects

with nocturnal myoclonus, and 2 alcoholics with sleep

apnea. The remaining sample was comprised of 31 men:

control subjects (n ¼ 15) and alcoholic patients (n ¼ 16).

Alcoholic patients were hospitalized for 2 weeks in theAlcohol and Drug Treatment Program at the Veterans

Affairs San Diego Healthcare System (VASDHS) before

testing in the current research study. Nonpatient con-

trols were identified though a standardized search

strategy of the San Diego area involving the placement

of flyers and advertisements in local newspapers. In

addition, a direct mailing to the San Diego County

veterans population was used to target controls whowere age matched (�5 years) and had sociodemographic

characteristics similar to alcoholic patients. After com-

plete description of the study, written informed consent

was obtained, using procedures approved by the Human

Subjects Protection Committees at University of Cali-

fornia, San Diego, VASDHS, and University of Cali-

fornia, Los Angeles. Sleep data from a larger cohort of

alcohol dependent patients and controls have been re-ported previously (Irwin et al., 2000, 2002).

Research diagnoses of controls and alcoholic patients

were made following the administration of a semi-

structured interview developed by the multisite Collab-

orative Study on the Genetics of Alcoholism (Bucholz

et al., 1994). Control subjects fulfilled DSM-IV criteria

for Never Mentally Ill (American Psychiatric Associa-

tion, 1994). Alcoholic patients fulfilled DSM-IV criteriafor Alcohol Dependence that had occurred in the

absence of major pre-existing or concomitant psychiat-

ric disorders (American Psychiatric Association, 1994;

Schuckit, 1985) including secondary depression that

may confound interpretation of EEG abnormalities in

alcoholics (Gillin et al., 1990a,b). Severity of depressive

symptoms was evaluated on the day prior to beginning

the sleep protocol using the 24-item Hamilton Depres-sion Rating Scale (HDRS), a frequently used, inter-

viewer administered measure that shows good internal

consistency (Endicott et al., 1981).

All participants were in good health as determined by

medical history and laboratory screening blood tests. To

measure adiposity, subjects� height, and weight were

assessed using a balance beam scale. Body mass index

M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360 351

(BMI) was computed as weight, in kilograms, divided byheight, m2. None of the alcoholics had histories of overt

alcohol-related liver disease and no subject was taking

medications known to alter sleep wake activity (e.g., b-blockers, psychotropic medications) within 2 weeks of

EEG sleep assessment. Alcoholics were studied after

acute and subacute withdrawal symptoms had resolved;

two alcoholic patients had been treated with diazepam

30 days prior to study. Nursing observations and ran-dom urine substance screens were used to confirm ab-

stinence during treatment.

2.2. Procedures

Two weeks before entry into the study, sleep–wake

activity diaries were obtained in alcoholics and controls.

All subjects were sleeping regularly between 22:00 hours(h) and 07:00 h with similar average bed and wake times

in the controls (23:50 h/06:40 h) and alcoholics (23:12 h/

06:36 h). As previously reported (Irwin et al., 2002),

subjects participated in a five night protocol. The first

night involved adaptation to the sleep laboratory and

screening for sleep apnea and nocturnal myoclonus. The

second night involved an uninterrupted period of noc-

turnal sleep. On the third, fourth, and fifth nights, sleeprecordings were obtained along with serial blood sam-

ples. On the third night, the baseline night, subjects were

allowed to sleep from 23:00 to 06:30 h; lights out after

23:00 h. On the fourth night, PSD-E was administered

and subjects were kept awake from 23:00 to 03:00 h with

sleep time from 03:00 to 06:30 h. On the fifth night,

subjects were allowed uninterrupted recovery sleep from

23:00 h to voluntary awake time. The effect of sleepdeprivation on recovery night EEG sleep in alcoholics

vs. controls was previously reported (Irwin et al., 2002).

Blood sampling was done at 23:00, 03:00, and 06:30 h

via an IV catheter without waking the subjects (Redwine

et al., 2000, 2003). After the blood was obtained, it was

delivered into ethylenediaminetetraacetic acid contain-

ing tubes and placed on ice and immediately centrifuged

for acquisition of plasma. All samples were then storedat )80 �C until assay. Sleep EEG measures were ob-

tained during continuous polysomnography recordings

between 22:00 and 06:30 h during each of the experi-

mental nights and visually scored as previously de-

scribed (Rechtschaffen and Kales, 1968; Redwine et al.,

2000, 2003).

2.3. Cytokine and liver function assays

Plasma levels of IL-6 and TNF were quantified by

means of enzyme-linked immunosorbent assay methods

(R&D Systems, Minneapolis, MN). All samples from

control and alcoholics were assayed at the same time, in

a single run with a single lot number of reagents and

consumables employed by a single operator. The intra-

assay coefficients of variation for all variables were lessthan 5%. Repeated samples of IL-6 across the three

nights were available for 11 controls and 12 alcoholics;

whereas samples of TNF were available for 13 controls

and 14 alcoholics. Values of liver function tests were

obtained and measured by previously described methods

(Irwin et al., 1990).

2.4. Statistical analyses

Data were analyzed using SPSS version 11.5 for

Windows and missing values were substituted by sin-

gle-point multiple imputation using NORM version

2.03 (Schafer, 2000). Group differences in sociodemo-

graphic data, alcohol and drug consumption histories,

and liver function tests were tested using one-way

ANOVAs. To evaluate group and night differences insleep variables, repeated measures multivariate ANO-

VAs (MANOVAs) were employed, using a 2 (group:

alcoholic, control)� 3 (night: baseline, PSD-E, and

recovery) design. The dependent variables in the MA-

NOVAs included sleep variables across three broad

domains: sleep continuity, sleep architecture, and REM

variables. For those sleep domains in which group

main effects were found by the MANOVA, subsequentunivariate ANOVAs were performed on the individual

sleep variables. The impact of experimental sleep de-

privation on sleep measures are not presented in this

paper as they have been reported elsewhere (Irwin

et al., 2002).

To evaluate group, night and time differences in cir-

culating levels of proinflammatory cytokines, repeated

measures ANOVAs were run using a 2 (group: alco-holic, control)� 3 (time: 23:00, 03:00, and 06:30 h)� 3

(night: baseline, PSD-E, and recovery) design. Minor

violations of normality were present in these variables

but previous studies have shown that ANOVA is not

appreciably impacted by violations of this magnitude

(Tan, 1982). Significant interactions were followed-up

with examination of group and time effects within the

individual nights using group� time mixed designANOVAs. In addition, based on a priori hypotheses and

prior findings that alcoholics have disordered sleep and

a defect in the recovery of sleep following sleep loss,

planned comparisons were performed to test whether

proinflammatory cytokines were elevated in alcoholics

within each night of the study, and whether adminis-

tration of sleep deprivation led to greater nocturnal el-

evations of IL-6 and TNF in alcoholics as compared tocontrols. It should be noted that these planned com-

parisons do not require a significant omnibus F finding

in order to be evaluated appropriately. According to

Keppel (1991), an unprotected analytic comparison of

group differences only occurs when no a priori hypoth-

eses exist or a nonsignificant omnibus F finding is

obtained.

352 M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360

Regression models were used to test the contributionof alcohol dependence on mean nocturnal levels of

proinflammatory cytokines across all time points com-

pared to the relative contribution of sociodemographic,

clinical, and/or alcohol consumption variables that dif-

fered between the groups. Using backward regression

analyses, the following predictor variables were entered:

alcohol consumption, BMI, education, and control/al-

coholic status as a binary variable. The dependentvariables (IL-6 and TNF) in these regressions were

modestly skewed. Given that multiple regression is not

as robust to nonnormality as ANOVA, multiple re-

gressions were conducted with log transformed depen-

dent variables. Although it is often accurate to test such

influences with covariate ANOVAs, this was not ap-

propriate in the current study given the causal effects

of chronic alcohol use to reduce body fat mass (Nicolaset al., 1993; Santolaria et al., 2000), and the high

correlations between the covariates (e.g., alcohol con-

sumption and BMI, see below) (Overall and Woodward,

1977; Tabachnick and Fidell, 1996).

To evaluate the hypothesis that severity of disordered

sleep is associated with alterations of cytokines, re-

gression models were also used. These regression anal-

yses were restricted to the EEG sleep variables thatdiffered between alcoholics and controls and were fur-

ther focused on measures of sleep latency, delta sleep,

and REM sleep. Sleep latency and percentage of delta

and REM sleep during the first and second half of the

night were evaluated in relation to proinflammatory

cytokines at 23:00, 03:00, and 06:30 h. Prior evidence

has shown that these aspects of sleep are associated with

altered levels of IL-6 (Redwine et al., 2003; Spath-Schwalbe et al., 1998). Levels of IL-6, TNF, and sleep

latency showed a modest skewed distribution; thus,

values were transformed to natural log values as noted

above. The regression models were repeated for IL-6

and TNF and entered the following predictor variables:

plasma levels of proinflammatory cytokines at 23:00 h,

at 03:00 h, at 06:30 h, alcohol consumption, BMI, and

education.

Table 1

Age, education, severity of depressive symptoms, and alcohol consumption

Controls (n ¼ 15)

Mean SD

Age (years) 39.5 8.8

Education (years) 14.5 1.6

Depressive symptoms (HDRS Scores)a 1.5 1.9

Body mass indexb 28.0 4.7

Alcohol consumption (last 3 months)

Drinking days/month 5.3 10.1

Drinks/day 1.2 1.0

Days since last drink 109 160.0

aHRSD, Hamilton depression rating scale.b Body mass index, weight in kilograms/height in m2.

3. Results

3.1. Demographic and clinical characteristics

Table 1 shows that the two groups were similar in

age. However, as compared to the controls, the alco-

holics had less years of education and a lower BMI.

None of the subjects showed evidence of depression as

measured by HDRS scores or by meeting DSM-IV cri-teria for Major Depressive Disorder. As expected, the

alcoholics had more drinking days per month, had

drank more drinks per day, and had used alcohol more

recently than the controls. A small number (n ¼ 4) of

participants in the control group reported using alcohol

(1–2 drinks) in the week prior to onset of the sleep

protocol; however, all other controls did not report use

in the last 2 weeks and none of the controls used alcoholwithin 3 days of participating in the study. None of the

31 participants fulfilled criteria for primary substance

dependence, and other substance use was infrequent in

the alcoholics with use of marijuana (n ¼ 5), psycho-

stimulants (n ¼ 9), sedatives (n ¼ 1), occurring in only a

subgroup of alcoholics in the last 3 months. None of the

alcoholics had used substances in the last 2 weeks, and

none of the controls reported substance use. Values ofthe laboratory tests of liver function did not differ be-

tween the alcoholics and controls and were within the

normal range for all but two alcoholics who had modest

elevations above normal (data not shown).

3.2. EEG sleep

Consistent with our prior findings (Irwin et al., 2000,2002), alcoholics showed disturbances of sleep continu-

ity and sleep architecture as compared to the controls

(Table 2). For the sleep continuity variables (total sleep

time, sleep efficiency, and sleep latency), an overall

MANOVA found a significant group effect:

F ð3; 27Þ ¼ 3:4, p < :05. Subsequent univariate ANO-

VAs found that sleep latency was prolonged in the al-

coholics: (F ð1; 29Þ ¼ 8:7, p < :01). Total sleep time and

histories in control and alcoholic groups

Alcoholics (n ¼ 16) Alcoholism effect (df ¼ 1; 30)

Mean SD F p

41.6 6.6 0.57 .46

12.8 1.0 12.7 .001

1.5 1.6 .01 .92

24.1 2.3 8.7 .006

26.9 5.7 54.5 <.001

16.2 13.6 18.1 <.001

18.8 8.9 5.1 .03

Table 2

EEG sleep continuity and sleep architecture measures in control and alcoholic groups

Base line Partial sleep deprivation Recovery

Controls

(n ¼ 15)

Alcoholics

(n ¼ 16)

Controls

(n ¼ 15)

Alcoholics

(n ¼ 16)

Controls

(n ¼ 15)

Alcoholics

(n ¼ 16)

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Sleep continuity

Total sleep time (min) 354.3 69.2 339.6 49.2 175.9 34 177.7 14.7 379.3 52.1 385.9 30.5

Sleep efficiency (%) 84.2 12.1 78.3 10.3 88.8 5.9 86.4 5.4 87.6 10.3 84.8 5.4

Sleep latency (min) 10.0 11.4 29.3 29.4 7.4 3.3 12.3 9.6 8.6 6.9 20.7 18.6

Sleep architecture

Stage 1 (%) 5.7 3.5 9.1 3.2 4.7 3.5 6.5 3.4 6.1 3.4 9.7 3.8

Stage 2 (%) 69.4 7.7 65.6 5.8 64.2 9.0 64.5 9.3 65.8 9.7 64.3 5.3

Stage 3 (%) 3.5 4.5 1.8 1.9 8.2 7.8 2.9 6.6 6.4 6.7 1.5 1.5

Stage 4 (%) 1.1 1.7 0 0 0.8 1.5 0.2 0.4 1.5 2.8 0.1 0.2

Delta (%) 4.6 5.9 1.8 1.9 9.0 9.0 3.1 7.0 7.9 9.3 1.6 1.6

REM (%) 20.6 6.3 23.5 6.7 22.1 4.7 25.9 7.7 20.2 4.7 24.5 5.6

REM measures

Latency (corrected) 74.6 28.5 52.7 35.5 42.0 23.2 39.1 20.8 73.9 37.7 56.5 30.4

Density 1.2 0.9 1.5 0.9 1.3 .7 1.7 .8 1.4 1.0 1.6 0.8

Duration (1st period) 14.4 7.7 16 9.1 24.8 13.6 24.7 15.5 22.5 11.9 16.7 8.7

M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360 353

sleep efficiency were similar in the two groups. For the

sleep architecture domain (percentage of time spent in

stages 1, 2, 3, 4, and REM), an overall MANOVA also

showed a significant group effect: (F ð5; 25Þ ¼ 4:1,p < :05). Alcoholics showed differences from controls in

the following specific analyses: greater percentage of

Stage 1 sleep, F ð1; 29Þ ¼ 5:8, p < :05; lower percentage

of stage 4 sleep, F ð1; 29Þ ¼ 4:7, p < :05; and increases inthe percentage of REM sleep, F ð1; 29Þ ¼ 4:4, p < :05.For the REM measures (REM latency, REM density,

and REM duration), the overall MANOVA showed no

group differences.

3.3. Proinflammatory cytokines: responsivity during sleep

deprivation

Across the three nights, omnibus F test results indi-

cated that circulating levels of IL-6 were higher in the

alcoholics as compared to the controls with a group

effect of F ð1; 21Þ ¼ 3:7, p ¼ :07 (see Fig. 1). IL-6 sig-

nificantly increased across the nocturnal period with a

time effect of F ð2; 42Þ ¼ 6:8, p < :01, but there was no

main effect for night. No significant interactions for

group� time, group� night, night� time or group-� time� night were found for IL-6. To evaluate the a

priori hypothesis that sleep deprivation would lead to

nocturnal elevations of IL-6 in alcoholics as compared

to controls, a series of planned group comparisons were

tested across baseline, PSD-E, and recovery nights.

During the baseline night, alcoholics and controls had

similar nocturnal levels of IL-6. In contrast, during the

PSD-E night, nocturnal levels of IL-6 were significantlyhigher in alcoholics than controls, F ð1; 21Þ ¼ 4:4,p ¼ :05. During the recovery night, there was a group-

� time interaction for IL-6 in which alcoholics showed

exaggerated increases of IL-6: F ð2; 42Þ ¼ 3:9, p < :05.Increases of IL-6 from 23:00 to 3:00 h were found in the

alcoholics (F1;21 ¼ 10:4, p < :01) whereas the controls

did not change.

Similar to the findings for IL-6, omnibus F test results

indicated that circulating levels of TNF were higher in

alcoholics as compared to controls with an overallgroup effect of F ð1; 25Þ ¼ 5:8, p < :05 across all three

nights (see Fig. 2). Likewise, TNF significantly increased

across the nocturnal period obtaining a time effect of

F ð2; 50Þ ¼ 12:3, p < :001, similar to the nocturnal in-

creases for IL-6. There was no night effect for TNF.

Finally for TNF, there were trends for the following

interactions: group� time, F ð4; 100Þ ¼ 2:8, p ¼ :07;group� night, F ð4; 100Þ ¼ 3:1, p ¼ :05; and night-� time, F ð4; 100Þ ¼ 2:4, p ¼ :06. No group� time� -

night interaction was found for TNF. To evaluate the a

priori hypothesis that sleep deprivation would lead to

nocturnal elevations of proinflammatory cytokines in

alcoholics as compared to controls, a series of planned

group comparisons were done for TNF across baseline,

PSD-E, and recovery nights along with follow-up anal-

yses of the significant interactions. During the baselinenight, alcoholics had higher levels of TNF as compared

to controls: F ð1; 25Þ ¼ 6:1, p ¼ :02, but no change over

time. For the PSD-E night, alcoholics had higher levels

of TNF as compared to controls: F ð1; 25Þ ¼ 6:5,p < :05, and showed an increase of TNF from 23:00 to

03:00 h, F ð1; 25Þ ¼ 7:9, p < :01. Controls did not

change. During the recovery night, a nearly identical

pattern of results was found with alcoholics showingelevated levels of TNF with a group effect of

F ð1; 25Þ ¼ 4:5, p < :05, and an increase from 23:00 to

Fig. 1. Change of plasma levels of IL-6 (means� SEM) over the night during baseline, partial sleep deprivation, and recovery nights in alcoholics vs.

controls.

Fig. 2. Change of plasma levels of TNF (means� SEM) over the night during baseline, partial sleep deprivation, and recovery nights in the alcoholics

vs. controls.

354 M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360

03:00 h during the nocturnal period of F ð1; 25Þ ¼ 10:6,p < :001, not found in the controls.

Taken together, sleep deprivation is associated with a

differential responsivity of proinflammatory cytokines in

the alcoholics as compared to controls. As compared to

controls, alcoholics show elevated nocturnal levels of IL-6

only after administration of sleep deprivation. For TNF,

alcoholics showed elevated nocturnal levels during eachof the nights. Following sleep deprivation, nocturnal in-

creases of TNF occurred in alcoholics but not in controls.

3.4. Examination of confounding variables related to

alcohol dependence and proinflammatory cytokines

Several clinical variables may be related to the effects

of alcohol dependence on proinflammatory cytokines.Group differences in IL-6 and TNF could be related to

characteristic variables such as obesity, socioeconomic

status as estimated by education level, and alcohol con-

sumption. Obesity, for example, is associated with higher

levels of IL-6 and TNF (Strawbridge et al., 2000).

However, in this study, alcoholics showed lower BMI,

consistent with undernutrition and reduced body fat

mass that frequently occurs with chronic alcohol use

(Nicolas et al., 1993; Santolaria et al., 2000), and BMI

was negatively correlated with drinking days/month

(r ¼ �:52, p < :01) and average drinks per day

(r ¼ �:41, p < :05). Thus, to test the contribution of al-cohol dependence group status to IL-6 and TNF relative

to clinical variables such as alcohol consumption, BMI,

and education, multivariate analyses were conducted.

For average IL-6 across the nocturnal sleep protocol,

backward elimination regression analyses found that

alcohol dependence was associated with higher IL-6

levels (b ¼ :40, p ¼ :06), whereas all of the other vari-

ables were eliminated from the model. For average TNFacross the sleep protocol, alcohol dependence (b ¼ 0:87,p < :01) and education (b ¼ 0:68, p < :05) were associ-

ated with higher TNF levels but neither alcohol con-

sumption nor BMI were significant predictors.

M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360 355

3.5. Proinflammatory cytokines: associations with sleep

A further hypothesis of this study was that circulating

levels of IL-6 and TNF would temporally correlate with

measures of sleep continuity and architecture. A series

of regression analyses were conducted for each night to

evaluate whether disturbances of sleep were associated

with consequent nocturnal increases of IL-6 and TNF,

or whether abnormal elevations of these cytokinestemporally antecede disordered sleep. In the regression

model, the dependent variables were EEG sleep vari-

ables (sleep latency, and percentages of delta- and REM

sleep in the first and second half of the night) which

differed between the groups, with IL-6 or TNF at 23:00,

03:00, or 06:30 h, and alcohol consumption, BMI, and

education serving as predictors.

3.5.1. Interleukin-6 (23:00, 03:00, and 6:30 h): associa-

tions with sleep

Backward elimination regression analyses indicated

that IL-6 at 23:00 h was a significant predictor of sleep

latency for each of the nights: baseline, PSD-E and re-

covery. In addition, for the baseline and PSD-E nights,

education was also significant (Table 3). Neither IL-6 at

03:00 h, IL-6 at 06:30 h, nor alcohol consumption wasassociated with sleep latency. Fig. 3 illustrates the cor-

relations between plasma levels of IL-6 at 23:00 h and

sleep latency during the baseline (r ¼ :37, p ¼ :08), PSD-

E (r ¼ :55, p < :01), and recovery nights (r ¼ :55,

Table 3

Results of backward elimination regression analyses predicting sleep with IL

Baseline night PSD

Predictor B Predi

IL-6

Sleep latency IL-6 (23:00 h) .38� IL-6 (

Education ).58�� Educa

Delta sleep (%)

1st half, early night NS NS —

2nd half, late night NS NS NS

REM sleep (%)

1st half, early night NS NS —

2nd half, late night IL-6 (03:00 h) .53�� IL-6 (

TNF

Sleep latency Education ).50� NS

Delta sleep (%)

1st half, early night NS NS —

2nd half, late night NS NS BMI

REM sleep (%)

1st half, early night NS NS —

2nd half, late night TNF (06:30 h) .59�� TNF

* p < :05.** p < :01.*** p < :001.

p < :01) with similar correlations within the separategroups of alcoholics and controls.

Percentage of delta sleep either during the first or

second halves of the night were not associated with IL-6

during the baseline, PSD-E, and recovery nights. Like-

wise, for percentage of REM sleep during the first half

of the night, no significant predictors were found.

However, for percentage of REM sleep during the sec-

ond half of the night, circulating levels of IL-6 at 03:00 hwas retained as the only significant variable in the re-

gression model across all three nights (Table 3). Fig. 4

illustrates the correlations between plasma levels of IL-6

at 03:00 h and percentage of REM sleep in the second

half of the night (03:00–06:30 h) during the baseline

(r ¼ :53, p < :01), PSD-E (r ¼ :41, p ¼ :05), and recov-

ery nights (r ¼ :68, p < :001) with similar correlations

within the separate groups of alcoholics and controls.Neither IL-6 at 23:00 h, IL-6 at 06:30 h, alcohol con-

sumption, BMI, nor education predicted percentage of

REM sleep during the second half of the night.

3.5.2. TNF (23:00, 03:00, and 06:30 h): associations with

sleep

Backward elimination regressions using TNF at

23:00, 03:00, or 06:30 h, alcohol consumption, BMI, andeducation as predictors indicated that only education

was associated with sleep latency during the baseline

night (Table 3). Furthermore, none of the predictors

were associated with sleep latency during the PSD or

-6 and TNF measures

night Recovery night

ctor B Predictor B

23:00 h) .50�� IL-6 (23:00 h) .55��

tion ).40�

— NS NS

NS NS NS

— NS NS

03:00 h) .41� IL-6 (03:00 h) .68���

NS NS NS

— NS NS

).49� NS NS

— NS NS

(23:00 h) .62�� TNF (06:30 h) .55��

Education ).46�

Fig. 4. Associations between IL-6 at 03:00 h and percentage of REM sleep in the second half of the baseline night (r ¼ :53, p < :01), PSD-E night

(r ¼ :41, p ¼ :05), and recovery night (r ¼ :68, p < :001).

Fig. 3. Associations between IL-6 at 23:00 h prior to sleep onset and sleep latency in the baseline night (r ¼ :37, p ¼ :08), PSD-E night (r ¼ :56,

p < :01), and recovery night (r ¼ :53, p < :01).

356 M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360

recovery nights, in contrast with consistent relationship

between IL-6 at 23:00 h and sleep latency.

For the percentage of delta sleep during the first- and

second halves of the night, none of the predictors were

significant in the regression models in any of the nights

except for a relationship between BMI and delta sleep

during PSD-E night (Table 3). Likewise, for percentage

of REM sleep during the first half of the night, none ofthe predictors were significant across the three nights.

However, for REM percentage during the second half of

the night, circulating levels of TNF at 06:30 h was re-

tained in the regression model for the baseline and re-

covery nights. Taken together, the associations between

the two proinflammatory cytokines and REM sleep

suggest that nocturnal IL-6 at 03:00 h predicts increases

of REM sleep which in turn may have consequences formorning levels of TNF.

4. Discussion

This is one of the first studies to show that abstinent

alcohol dependent persons show elevated circulating

levels of IL-6 and TNF, independent of the relative

contribution of confounders. In addition, this study in-

dicates that disordered sleep promotes nocturnal in-

creases in these proinflammatory cytokines. Following

experimental sleep deprivation, alcoholics show abnor-

mal elevations of IL-6 and TNF. Alternatively, such

nocturnal elevations of IL-6 may have a negative influ-

ence on sleep amounts or depth, as alcoholics show adefect in the recovery of delta sleep following sleep de-

privation (Irwin et al., 2002).

Consistent with the notion that cytokines have re-

ciprocal effects on sleep, this study provided evidence

that physiological elevations and individual differences

in cytokines temporally predict sleep continuity and

architecture. By temporally examining the expression of

IL-6 and TNF across the night in association with sleeplatency, this study found that elevated levels of circu-

lating IL-6 prior to sleep onset predicted prolonged sleep

latency. This association was reliably demonstrated

across three separate nights relative to the independent

contribution of alcohol consumption, BMI, and educa-

tion. The direction of the temporal associations between

IL-6 and sleep latency was confirmed by the time course

M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360 357

of assessment of cytokines in relation to sleep; there wasno association between measures of sleep latency and

subsequent elevations of IL-6 or TNF. Since alcohol

consumption severity was not related to sleep latency, it

is possible that nonalcohol-dependent clinical popula-

tions who show elevated levels of IL-6 may also be at

risk for prolonged sleep latency. Collectively, these data

argue against the notion that IL-6 has sleep-initiating

effects which has been proposed on the basis of studiesin patients with excessive sleepiness (Vgontzas et al.,

1997, 1999). Rather, the current findings are consistent

with the previously suggested roles of IL-6 in the phys-

iological regulation of sleep; namely, increases of IL-6

lead to sleep arousal and fragmentation (Hogan et al.,

2003). However, it is also possible that the IL-6 changes

serve as a marker for other biological alterations (e.g.,

cortisol) which are associated with EEG sleep (Spath-Schwalbe et al., 1998).

In addition to the effects of IL-6 on sleep latency,

higher levels of nocturnal levels of IL-6 (at 03:00 h) was

correlated with larger percentages of REM sleep during

the second half of the night, during which time REM

sleep is most prominent. Furthermore, alcoholics had

significantly more REM sleep percentage than controls,

and amounts of REM sleep predicted morning levels ofTNF. Prior observations from our laboratory have

found that circulating concentrations of IL-6 are ele-

vated during periods of REM sleep (Redwine et al.,

2000). In contrast, stimulated production of the anti-

inflammatory cytokine IL-10 was found to predict delta

sleep (Redwine et al., 2003). Taken together, the present

temporal correlations further suggest that physiological

elevations of IL-6 and possibly other proinflammatorycytokines may be involved in inducing abnormal

amounts of REM sleep in alcoholics, with implications

for populations who have elevated levels of IL-6 (e.g.,

aged persons, depressives, and patients with inflamma-

tory disorders) (Kiecolt-Glaser et al., 2003; Musselman

et al., 2001; Zautra et al., in press). Experimental con-

firmation of this hypothesis is needed, possibly by the

administration of specific cytokine antagonists.We speculate that these findings linking IL-6 to dis-

ordered sleep extend well beyond alcohol dependence.

Recent epidemiological evidence has shown that sleep

disturbance is an independent predictor of mortality

(Dew et al., 2003). In healthy older adults, prolonged

sleep latency (comparable to that found in our alcoholic

sample) yielded a 2-fold elevated risk of death. Further-

more, increases of REM sleep>25.7% (also similar to theamounts found in the alcoholics) led to 1.7 times greater

risk of death. In addition, IL-6 is associated with a variety

of illnesses (Ridker et al., 2000; Zautra et al., in press),

and elevations of this cytokine above 3.19 pg/ml lead to a

2-fold greater risk of death in older adults (Volpato et al.,

2001). Importantly, average nocturnal levels of IL-6 ex-

ceeded this threshold in alcoholics and were further ex-

acerbated following sleep deprivation. In other chronicdisease conditions where immune activation is provoked,

it is also possible these patients also may be at risk for

disordered sleep with further health consequences.

In concert with sleep loss and disordered sleep, ab-

normal elevations of IL-6 and TNF may have additional

behavioral consequences. For example, given the re-

ciprocal interactions between sleep and proinflamma-

tory cytokines, an important question is whether thesecytokine mechanisms contribute to the persistent se-

verity of sleep disturbance in recovering alcoholics. Such

a scenario would suggest that modest sleep loss, as

shown here, leads to increases of proinflammatory cy-

tokines which in turn delays sleep onset and further

perpetuates an abnormal cycle of proinflammatory cy-

tokine expression along with disordered sleep.

In addition, peripheral proinflammatory cytokinesare capable of exerting direct effects on central nervous

system function. Bower and colleagues (2002) have

found that immune activation is associated with severity

of fatigue symptoms in women recovering from breast

cancer, and Capuron et al. (2002) suggest that immune

activation is associated with cognitive impairments.

Likewise, experimental immune activation results in in-

creases of anxiety and depressed mood, which are cou-pled with declines of memory functions (Reichenberg

et al., 2001). Whether disordered sleep and daytime

elevations of proinflammatory cytokines contribute to

depressive symptom severity and cognitive disturbances

in recovering alcoholics is not known.

The present data have been gathered under natural-

istic conditions where changes in cytokine levels are

likely to have been present for days to months, ratherthan minutes to hours as with acute challenge studies.

Thus, in contrast to the present findings, Spath-Schw-

albe et al. (1998) found that low exogenous doses of IL-6

suppressed REM sleep in humans within 4 h of admin-

istration. Whereas acute cytokine-induced HPA axis

activation was thought to mediate suppression of REM

sleep (Spath-Schwalbe et al., 1998), we have found that

the nocturnal secretory profile of cortisol is similar inalcoholics and controls, making it unlikely that the as-

sociation between IL-6 and REM sleep is due to HPA

axis activation (Irwin et al., 2003). Similarly, Mullington

et al. administered an acute dose of endotoxin immedi-

ately before ‘‘lights off’’ and found that nonREM sleep

was enhanced along with increases of proinflammatory

cytokines (Mullington et al., 2000), but higher doses of

endotoxin disrupted sleep. Likewise, in animals, acutecentral doses of proinflammatory cytokines enhance

nonREM sleep and suppress REM sleep (Opp and

Toth, 2003). Thus, as compared to the present study,

acute experimental studies yield a different profile of

findings possibly due to a different time course of action

and/or the induction of higher levels of cytokines than

found in alcohol dependence.

358 M. Irwin et al. / Brain, Behavior, and Immunity 18 (2004) 349–360

With peripheral blood sampling, Haack and col-leagues (2000, 2002) have recently suggested that diurnal

variations and nocturnal increases of IL-6, but not

TNF, are confounded by the placement of an intrave-

nous catheter which leads to local release of IL-6 over

several hours and linear increases in levels of IL-6 de-

tected in peripheral blood (Haack et al., 2000, 2002).

However, Born et al. found no change in circulating

levels of IL-6 over the course of 24 h blood sampling(Born et al., 1997). Likewise, in hourly sampling from an

indwelling catheter, Redwine and colleagues found that

circulating levels of IL-6 were unchanged over 5 h of

wakefulness (Redwine et al., 2000). Moreover, following

the onset of sleep, a stable plateau of IL-6 is maintained

over 8 h (Redwine et al., 2000). Finally, we have found

that intracellular IL-6 and TNF expression is unchanged

over 24 h following placement of an intravenous cathe-ter. Importantly, in the present study, the procedures for

placement of an intravenous catheter were the same for

both groups and similar across each of the nights,

making it unlikely that placement of an intravenous

catheter substantially accounted for group differences of

IL-6 and TNF or the differential effect of sleep loss and

recovery sleep on IL-6 levels in the alcoholics as com-

pared to the controls.Limitations of this study include small sample size

and gender, and it is not known whether these findings

generalize to other community samples or to female

alcoholic patients. In addition, an African American

population was exclusively studied. Nevertheless this

population warrants continued investigation as we have

found that African American alcoholics have greater

disturbances of sleep (Irwin et al., 2000), a defect in thehomeostatic regulation of sleep (Irwin et al., 2002), and

abnormal expression of pro- and anti-inflammatory

cytokines with consequences for innate and adaptive

immune responses (Irwin and Miller, 2000; Redwine

et al., 2003) as compared to European American alco-

holics. Together, these changes might contribute to the

disparities in health and increased morbid outcomes in

African American alcoholics as previously reviewed(Irwin and Rinetti, in press). Finally, the sleep protocol

focused on sleep loss during the early part of the night, a

time of low propensity for REM sleep. Timing of sleep

deprivation with respective effects on cytokine expres-

sion warrants further consideration.

The results of this study indicate that activation of the

immune system is associated with prolonged sleep la-

tency and increases of REM sleep. Given other data thatabnormal sleep continuity and REM sleep amounts

predict an excess risk of dying (Dew et al., 2003), this

study represents a further step in understanding the

mechanisms that link disordered sleep to mental and

physical health outcomes in alcoholics with implications

for other clinical populations with chronic inflamma-

tion. Finally, the contribution of cytokines in the regu-

lation of sleep suggests that cytokine mechanisms are apossible target for interventions to optimize sleep initi-

ation and sleep quality in alcoholic and other at-risk

populations.

Acknowledgments

This work was supported in part by Grants

AA10215, AA13239, DA16541, MH55253, AG18367,

T32-MH19925, AR/AG41867, and M01-RR00865,

General Clinical Research Centers Program, and theCousins Center for Psychoneuroimmunology. The au-

thors thank Dr. Jason Cole for consultation regarding

the statistical analyses.

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