Sleep vs. Cellular Immune

8/2/2019 Sleep vs. Cellular Immune

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Partial night sleep deprivation reduces natural k illerand cellu lar immune responses in humans

M ICHAEL IRW IN,1 JOHN MCCUNTICK , CAROLYN COSTLOW , MEUSSA FORTNER , JACK W IHTE ,AND J. CHRISTIAN GILLIND epartm ents of P sychiatry , U niversity of C alifornia, and San D iego V eterans A ffairs M edical C enter, S an D iego,C alifornia 92161, U SA

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0892-6638/96/001 0.0643/$01 .50 FASEB 64 3

ABSTRACT Prolonged and severe sleep depriva-tion is associated w ith alteration s of natural andcellu lar immune function. To determ ine whetheralterations of immune function also occur after evena modest loss of sleep, the effects of early-nightpartial sleep deprivation on circu lating numbers ofwhite b lood cells, natural killer (NK ) cell number andc yt ot ox ic ity , ly mp ho kin e- ac tiv at ed killer ([AK ) cellnumber and activity, and stim ulated interleukin-2(IL-2) production were studied in 42 medically an dpsychiatr ica lly healthy male volunteers. A fter a nightof sleep deprivation betw een 10 P .M . and 3 A .M ., areduction of natural immune responses as m easuredby NK cell activity , NK activ ity per number of NKcells, LAK activ ity, and LAK activity per num ber of[AK precursors (CD16,56, CD25) was found. Inaddition, concanavalin A -stimulated IL -2 productionwas suppressed after sleep deprivation due tochanges in both adherent and nonadherent cell popu-lations. A fter a night of recovery sleep , NK activityreturned to baseline levels and IL-2 production re-m ained suppressed. These data implicate sleep in themodulation of immunity and demonstrate that evena modest disturbance of sleep produces a reductionof natural immune responses and T cell cytok ineproduction.-Irwin, M ., M cClintick, J ., Costlow , C .,Fortner, M ., W hite, J., Gum, J. C . Partial night sleepdeprivation reduces natural killer an d cellu lar im -mune responses in humans. FASEB J. 1 0, 6 43-653(1996)Key Words: immunity natural killer cell aclivily interleu kin-2

SLEEP DISTURBANCE IS BELIEVED TO adversely affect hostresistance to infectious disease (1 , 2). Epidem iologic datashow that circadian shift workers who commonly experi-ence disordered sleep exhibit depressed cellu lar immunefunction and increased rates of respiratory tract infections(3 , 4). In animals, sleep deprivation lasting only 7 h im-pairs influenza viral clearance and specific influenza an-tibody production (5, 6), and sustained sleep loss has alethal outcome due to system ic infection and septicem iaw ith opportunistic bacterial m icroorganism s (7).Human studies involving prolonged sleep loss indicate

alterations of immune function . However, both enhancing

and suppressive effects on natural killer (NK )2 activ ityand on lymphocyte responses have been reported . For ex-ample, D inges and colleagues (8) found in 20 subjectsthat 64 h of sleep deprivation was associated w ith leuko-cytosis and increased NK cell activity, but w ith nochanges in lymphocyte counts or nonspecific pokeweedm itogen, phytohemagglutin in (PHA), or concanavalin A(Con A) lymphocyte proliferation. In contrast, Moldofskyet al. (9) reported in 10 men that 40 h of sleep loss in-duced a prolonged decline in NK activity and a delayednocturnal rise in lymphocyte proliferation to pokeweedm itogen but not to PHA . Finally , Palmblad and col-leagues (10) found decreased PHA-induced lymphocyteproliferation after 4 .8 h of wakefulness in 12 men and in-creased interferon production and decreased phagocyticactiv ity in 8 women deprived of sleep for 77 h (11).These data indicate a relationship between sleep and

immune function. However, healthy persons, stressed in-dividuals, or even depressed patients only rarely experi-ence to tal sleep loss throughout the night (12, 13), andthe effect of loss of sleep during only part of the night onnatural or cellu lar immune responses has been relativelyunexplored . Recently , w e reported that sleep deprivationin the late part of the night (wake tim e from 3 A .M . to 7A.M.) produced about a 30% decrement of NK activ ity in23 healthy men (14).The present study extended our previous findings on

late-night sleep deprivation and evaluated whether loss ofsleep in the early part of the night had sim ilar effects onnatural cyto toxicity . Furthermore, because there are in-consistent reports on the effects of sleep loss on NK ac-tivity due possib ly to competing alterations in cellularactivation and in the relative distribution of NK cells inperipheral b lood (8, 9), the simultaneous assessment of

hf whom correspondence and repr int r eq ue st s s ho uld b e a dd re ss ed ,a t: D e pa rtme nt o f Psychiatry Vi 16A , 3350 La Jolla Village D r., San D iegoV A M ed ic al C en ter, S an D ie go, C A 9 21 61, U SA .

2Abbreviations: ANO VA, analysis of variance; PBS, phospha te -bu f f-e re d s alin e; C on A , c on can ava lin A ; N K, n atu ral k ille r; L AK , lym pho k-in c- ac tiv at ed k ille r; I L- 2, in te rle uk in -2 ; P SD- E, e ar ly -n ig ht p ar tia l s le epdeprivat ion; M H- CR C, M en ta l H ea lt h C li ni ca lR es ea rc h C en te r; P II A,phytohemagglutin in; DM E, Dulbeccos m od i fi ed E ag l e; C BC , c om pl e te1)100(1count.


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cell activity, number of NK cells, activ ity per cell, andthe ability of NK cells to form lymphokine-activated kil-ler (LAK) cells w as obtained to provide detailed insightinto sleep regulation of this immune cell. F inally , the ef-fects of partia l night sleep loss on stimulated productionof in terleukin-2 (IL-2) was exam ined w ith further testingto determ ine whether alterations of both T helper and/ormonocyte populations accounted for suppression of thiscytokine after sleep loss.

METHODSSubjectsMa le v o lu n te er s (n , 46 ) w ere selected u sin g recruitment procedures ofthe Menta l Health Clin ical Research Center (MH-CRC) that involved as ta nd ard iz ed s ea rch stra te gy o f th e S an D ie go a re a u sin g a dve rtis em en tsin local new spa pers and university ne wsle tters. B efore givin g in form edconsen t for the present study, the volunteers underwent a rigorous psy-chiatric and medical evaluation by M H-CRC psychiatric research fel-low -physicians that inc luded psychiatric an d m edical histories, ph ysica l

exam ination, screening laboratory exam ination (chem istry panel, com -plete blood cell coun t, thyroid function tests, H IV test), and a formalstructured psychiatric diagn ostic in terv iew using the Schedule for Clin i-cal Interv iew (DSM-II1-R) (15). M ter consensual diagnosis by at leastthree M H-CRC psychiatrists, all subjects were found to have no l i fetimehistory of a DSM-III-R mental disorder such as major depression ors ubs tan ce d ep end enc e. M edic al h is to ry, ph ys ic al e xam in atio n, a ndla bo ra to ry te sts rev eale d th at the m en were in good m ed ic al h ea lth ;s ubje cts ha d n eith er h is to rie s of rec ent (< 1 0 da ys ) v iral in fe ction s n orhistor ies of d iseases (e.g., autoim mune disorders or cancer) that couldaffect immune function. In addition, none of the men reported usingpsychotropic m edications or other m edications such as prostagland ininhibitors (i.e., aspirin or nonsteroidal anti-inflam matory agents) orblockers known to affect sleep structu re and/or im mune function w ithina 10-day period before enrollment in the study. Screening laboratorytests including complete blood cell count, chem istry panel, liver andthyro id function, and H IV tests w ere w ith in norm al lim its. T his studywas lim ited to men because NK activ ity ha s been show n to fluctu ateacross the m enstrual cycle, which m ight alter interpretation of changesin NK activity during base line and the 5-night sleep. protocol (H . Coverand M . Irw in, unpublished data).O f the 46 subjects who were diagnos-tically and m edically evalua ted, fulfilled the specified inclusion criteria,and gave inform ed consent, 42 m en fu lly com pleted the study protocol;3 men were dropped because o f nocturna l myoclonus (tib ial index >

TABLE 1. Sleep measures during night s of baseline, early-n ight p ar tia l s le ep d ep riv ati on , an d recovery sleepBase li ne s leep n igh t,

mean (SD)P ar tia l s le ep d ep riv at io n n ig ht .

mean (SD)Recover y n igh t,

mean (SD)Significance

F PS leep cont in u it y

To tal sleep tim e (mm)S leep e f fi ci en c y (% )S le ep l at en cy (mm)

S l eep a rch it e ctu ren on-R EM sleep

(mm)Stage 1 sleep

(mm)(% )Stage 2 sleep

(mm)(% )

Stage 3 sleep(mm)(% )

S ta ge 4 s le ep(mm)(% )

Delta sleep(mm)(% )

REM measuresREM sleep

(mm)(% )

REM latency (mm)REM density (1st period)

3 92 .2 (4 2.7 )88.2 (6.9)1 5.8 ( 15 .1 )

3 25 .1 (3 8.7 )

3 6.6 ( 50 .8 )9 .5 ( 12 .9 )

2 29 .1 (3 4.1 )58.6 (7.6)

3 4.1 ( 20 .4 )8 .6 (5 .1 )

1 5.7 (1 6.8 )3 .9 (4 .1 )

4 9.8 (3 3.6 )1 2.5 (8 .3 )

8 5.5 (2 5.6 )2 1.6 (5 .4 )7 6 .6 ( 26 .9 )1.3 (0.6)

1 98 .4 * (1 7.1 )91.3 (7 .2)

8.4* (12.68)

1 64 .1 * (2 3.8 )

1 0.8* (6 .1)5 .5 (3 .1 )

1 01 .2 * (2 2.8 )5 1.3 * (1 2.1 )

25.9 * (1 5.3)1 3.0 * (7 .5 )

1 4.6 (1 8. 3)74* (9 .4)

40.5 (26.5)20.4 * (1 3.4)

457* (16.9)22.7 (7.8)

4 1. 1* ( 32 .8 )1.6 (0.7)

437.01 * (62.4)9 1.9 (1 1.4 )1 1.2 * (1 2.8 )

3 53 .1 (5 3.3 )

2 1.1 (6 .9 )4 .9 (1 .6 )

244.2 (49.8)5 6.5 ( 10 .5 )

43.8 (24.4)9 .9 (5 .3 )

24 .4* (28.8)53* (6.2)

68.2* (45.6)1 5.2 (9 .7 )

1 03 .4 ( 25 .4 )2 3.3 (4 .8 )7 3. 6 ( 33 . 2)1.5 (0.9)

363.93. 03. 4

233.5

5. 53. 1

217.510.1

11.38. 3

5. 64. 9

17.916.5

77.40. 919.72. 7

0.0010.060.04

0.001

0.010.06

0.0010.001

0.0010.001

0.010.01

0.0010.001

0.0010. 4

0.0010.11

P


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RES EARCH COMMUN IC AT IO N

SLEEP DEPRIVATION AND IMMUNITY 645

15), and 1 subject refused to undergo sleep deprivation during theexperim ental protocol. These 42 subjects had an average a ge o f 3 5. 0years (SD=11.1; range: 25 to 65 years) and had achieved a meaneducational level of 16 .1 years (SD1.8; range : 12 to 21 years). Th eethnic composition of the sample was d iverse w ith 83% white, 3%black,and 14% Asian ; m arita l characteristics of the sam ple showed that48% were sing le, 40% married, and 1 2% d iv or c ed /s ep a ra te d .

Substance use was infrequent in the sample: only two subjects werecurrent sm okers, and histories o f alcohol consum ption revealed an aver-age of 1.7 (sD=1.1; range: 0 to 5) drinks per drink ing day and 11.7(S 0 12.0; range : 0 to 60) total drinks per month. Last co nsum ption ofalcohol was on the average 20.9 (sD28.7) days before en try into thepresent study, w ith no other substance use (inc luding marijuana) re -ported w ithin the last m onth. F inally , s leep.-wake activ ity diaries ob -ta ined during the 2 wk before entry into the study confirmed that allvolunteersregularlywere sleeping nights between the hours of 10 P.M.and 7 AM., with reported average totalsleep time of 7.2 h ( SD =0 .7 ;range:6 to 9) per night.ProceduresThe study used a repeated measures w ithin sub jects design and evalu-ated differences in the im munologic variables between base line, im me-diately after early-n ight partia l night sleep deprivation (PSD-E ), andafter a n ight of recovery sleep . The assessment of immunity includedcomple te blood cell coun t and diffe rentia l, NK ce ll activity, LAK cellactivity,nd IL-2 p ro du ctio n a fte r s tim ula tio n wmth Con A. Assay of NKactiv ity was obtained in the total sample . Whether the effect of s leeploss on NK activity was due to 1) a lte ra tio ns in th e re la tiv e d is tr ib utio nof NK cells (CD16,56); 2) the state of NK cell activation (NK activ ityper NK cell); 3) the ability of IL-2 to activate NK cells (LAK cellactiv ity); and/or 4 ) changes in the production of IL-2 , a potent stim ula-tor of NK cell activ ity , was evalua ted in subgroups of the total sample.In addition, further substudies determ ined whether the reduction ofLAK activ ity was due to changes in the number o f LAK precursors(CD16,56 + CD25) and whether PSD-induced alterations of IL-2 pro-d uc t io n w e re m ed i at ed b y c ha n ge s i n n o na dh er e nt ( ly mp h oc yt e ) a n d /o radhe re n t (mono cy te ) c e ll p o pu la ti on s .

Ba seline evalua tion of the im mune va riables w as conducted within 2 w kbefore subject entry into the sleep laboratory and the sleep deprivationprotoco l. F or N K activity, m ultip le baseline d eterm inations w ere o btainedto provide an assessm en t of the stability of this im mun e m ea sure. Co nsis-tent w ith procedures proposed by Korn et a!. (16), m ultip le independentbaseline valu es, rather than a single value , can be m ore re liab ly com paredto a n in te rve ntio n va lu e fo r sta tistica l a na lys is a s w e ll a s f or th e id en tifica -tion of subject characteris tics that predict changes of these immunemeasures. For other m easures of im mun ity (i.e., LAK activ ity, T, NK, andLAK cell enumeration, and IL-2 production), which have coeffic ients ofvariationrangingfrom 5 to 12%, one baselinevaluewas obtained.

Mter evaluationof immune functionatbaseline,subjectsenteredthesleep deprivationprotocoland sleptin the sleep laboratoryfor 5 con-secutive nigh ts: night 1, adaptation ; night 2, baseline sleep; night 3,baseline sleep; night 4, PSD-E; a nd n ig ht 5 , r e co ve ry .O n t he n ig ht o fPSD-E, subjectswere admitted to thesleeplaboratoryat8 P.M. a nd w er ea sk e d t o s t ay a w a ke u nt i l3 AM.. Wakefulness w as m onito red by observ-ing the patient w ith a camera and by EE G r ec o rd in g s. F o r t he immu -n olo gic a ss es smento f w h ite b lo od c ell c ou nts , N K a ctiv ity , a nd IL -2production during the sleep laboratory protocol, sam ples w ere obtainedt he m or n in g a f te re it h er t h e first o r s ec ond baseline night in the sleepl ab or at or y t o e va lu at e w he th er s le ep in g i n t he l ab or at or y i nd uc ed an on sp ec if ic r ed uc ti on o f i mm un e f un ct io n (s leep base line), a fter thenightof early-n ight partia l s leep deprivation (PSD-E; awake tim e 10 P .M .to 3 AM.), and afterrecovery sleep.Assay of LA K activityand lympho-c yt e s ub se t e n u me ra ti o n w er e o bt a in ed a t b a s el in e an d a f te rP SD -E . O nbasel ine a s we ll a s experi mental days, b lood (45 m l) wa s collectedbetween 7 AM . and 9 AM . i n h ep ar in iz ed v ac ut ai ne r t ub es f ro m a na nt ec ub it al v ei n o f a s up in e s ub je ct r es ti ng f or a t l ea st 1 5 mm. Ina dd it io n t o t he se b lo od s am pl es , nocturnal b lo od s am pl in g u si ng a nindwelling intravenouscatheter(resultsto be reportedseparately)was

c on du c te d d u ri ng o n e o f t h e b a s el i ne a nd PSD-E nights.The cumulativeamount of b lood sampled from a subject did not exceed 500 m l duringthe sleep deprivationprotocol.

S le ep E EG m ea su re s w er e c on du ct ed t hr ou gh ou t t he s le ep d ep ri va -t i on p ro to co li n o r d er t o m ea su r e t h e e f fe c tso f P SD - E o n s le ep c on t in u-i ty a nd e va lu at e p os si bl e c ha ng es i n s le ep a rc hi te ct ur e du ri ng t herecovery sleep. Briefly, subjects s lept in individual bedrooms in theMH-CRC, and polygraph ic recordings of EEC, electro -oculogram(EOG ), and subm ental EM G were perform ed during the subjects regula rs le ep in g h ou rs b et we en 1 0 P.M . and 7 AM. for e ach of the 5 nights.D ur in g t he a da pt at io n n ig ht , r e co rd in gs o f o xy ge n d es at ur at io n a n dtib ia! m yoclonus were also obtained to exclude subjects w ith eithersleep apnea or nocturnal m yoclonus. Sleep data from the first night werenot used in the analyses. It was not necessary to immobilize the arm inwhich the indwelling catheter was placed, and this blood samplingprocedure was n ot foun d to alter s teep; sleep measu res in th e t wobase lin e nights w ere sim ilar, although sleep efficiency w as redu ced from88.6 7.8% to 83.5 13.0% (t=2.3, P< 0.05).

S le ep r ec or ds w er e v is ua ll y s co re d a cc or di ng t o t he c ri te ri ao fR ec ht sc ha ff en a nd K al es ( 17 ). Da ta f ro m e ac h 3 0 s e po ch w er e e nt er edi nt oa c om pu te r p ro gr am ( de ve lo pe d b y P . Clopton, S . C ol sh an , a nd L .W et he r el l, Sa n D ie g o V AM C) t h at ta ll i es h e s um ma r y s t at is t ic so r ea chsubject.Sleep onset was def ined a s t he f ir stm in ut e o f s t ag e 2 o r R EMs le ep f ol lo we d b y a t l ea st 8 mm of sleep in the next 9 mm. A REMperiod w as d ef in ed b y n ot l es s th an 3 c on se cu ti ve mm of R EM sleep.S le ep e ff ic ie nc yw as t he r at io of t ot als le ep t im e t o t he t im e b et we en go od n ig ht a nd go o d m o rn in g m ul t ip li e d b y 100. Sleep architecturewa s d efin ed a s th e d ura tio n o f time spent asleep in non-REM sleep,stages 1 through 4. REM density was an estimate o f the number of eyemoveme nts/mm o f REM sl eep,scor ed on a sca le of 0 t o4 per 30 s epochb ut e xp re ss ed o n a s ca le o f 0 t o 8/ mm . S le ep r es ea rc h te ch nicia ns w erer eg ul a rl y te s te d o n s c or in g r el i ab il it yn d h i gh s t an da r ds w er e m a in -ta ined: s leep latency (r0.96), REM latency (r0.99), REM density(r0 .91), amounts of stages 3 and 4 ( r0.85) , an d to talsleep t ime(r0.99).Immunologic assaysComplete blood countWhite blood count and differentialf granulocytes,lymphocytes, andmo nocyte s were p erform ed using stan dard me thods (S TKS, Co ulterC or p, H ia le ah , F la .) a t t he S an Diego VA Medical Center C lin icalLaboratory (S.Baird,Director).

L ym ph ocyte p rep aratio nPeriphera l blood lymphocytes were isolated using Leucoprep tubes(Becton-D ickinson, Franklin Lakes, N.J.), washed tw ice with phos-pha te-bu ffered saline (PBS) (G ibco Life Techno logies Inc., Grand Is-land, N .Y.), and resuspended in a 1:1 m ixture of RPM! 1640 an dDulbeccos m od ified Eagle (DM E) m ed ia supplem ented w ith 10% fetalc a lf se ru m ( H yc lo ne , L o ga n, U t ah ; i n ac ti v at ed1 h in 56 #{17 6}Ca te r b ath ),4 mM glutammne, and 20 mM HEPES f or c ell c oun tin g . T h is p ro cedur eyieldsa cellsuspension thattypicallycontains cellswith a viabilityf>98%.

NK activityBefore assay o f NK ac tiv it y, a dhe ren t c e lls w e re r emov ed by i ncuba ti onof mononuc learce l ls on 15 0 m m d ia me te r p la st icp et rid is he s f o r 1 h a t37#{176}Cn 5% CO2 in air. Nonadherent cells were removed by gentlew as he s w it h w ar m P BS c on ta in in g 5% f et alc al fs er um a nd r es us pe nd edat 2 x 10 6 cells/miin RPMI 1640 s up pl em en te d w it h 1 0% h ea t i na ct i-vated fetal calf serum , 4 mM glutam ine, and 20 mM HEPES buffer.E ffecto r cells were coincubated w ith 5tchrom ium (New England Nu-clear, Boston, M ass.) -la beled K562 targe ts cells at effector to target cellratios o f 40:1, 20:1, 10:1, and 5:1, and a standard 3 h chrom ium releaseassay was then performed in 0.2 m l volumes in U-bottom m icroplates.B riefly , plates were centrifuged for 2 mm at 500 x g, incubated for 3 h


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646 Vol. 10 April1996 T he F AS EB J ou rn al IR WIN E TA L.


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Figure 1. Effects of early-night partia l sleep deprivation on mean SC M to ta l w hite b lo od c ell c ou nt s, a nd p er ce nt ag ean d n um ber of gra nulocytes, lym phocytes, and m onocyles in 2 8 he althy m en. Re peated m easu res AN OVA over baseline,baseline sleep, PSD-E, and recovery demonstra ted no time effect for total white blood cell count (F0 .92; 3,81 ;P0.44). PSD-E was associated w ith a significant decrease of percentage, bu t not num ber o f granulocytes (F= 11.4,df3,81, P< 0 .0 01 ; 2 .2 . d f3 ,8 1, P0.09), and w ith a significant increase of perce ntage a nd num ber of lym phocytes(F 13 .1 , d f# {1 76 }3 ,8 1;< 0 .0 01 ; F 17 .1 , d f# {1 76 }# {1 76 }3 ,8 1,< 0.001). C om pared w ith b aseline values, pla nned com pa risonsdem onstrated change s in percenta ges of gra nulocytes and in pe rcentages and num bers o f lym pho cytes after PSD -E, butnot at other times. A significant increase of percentage and number o f monocytes (F4.0, 3,75, P< 0 .0 1; F # {1 76 }6 .4 ,df=3,75, P< 0.001) was also found across the 4 n ights, although p lanned com parisons found that this difference wasdue to increases be tween baseline and sleep EEC values and was not rela ted to the effects of PSD.


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A2 3 Baseline PSD-E Recowry

Basel ine Sleep

SLEEP DEPRIVATION AND IMMUNITY 647

RES EARCH COMMUN IC AT IO Nat 37#{176}Cn 5 % C O2 . a nd c en tr if ug ed ag ai n b ef or e h a rv es ti ng of 0 .1 m lsupernatant from each well. Radioactivity in the superna tant was deter-m ined by a gamma counter; the percentage of 51chrom ium released ateach effector to target cell ratio w as calculated to give percent of specificcytotoxic ity . Assay data were excluded on those experimental days inwhich spontaneous re lease of chrom ium was more than 10% of the totalre lease and/or no specific cytotoxic ity w as m easurable in the la boratorycon tro l t ha t was run on each assay date.

Data for NK activity are presented as the number of lytic units in 106cells (18). Lytic unit values reflect the re lative cytotoxic ity of the lym -phocyte preparatio n a nd a re m ore accurate than co mpara tive chro miumrelease data which may be influenced by the percent of lysis (18). Lyticun its were obtained by fitting scale fam ilies of curves to the data and inth is case each curve was fit separately to the modified exponentia lfam ily of equations. One lytic unit was defined as the num ber o f effectorcells killing 20% of the targe t cells.Ce ll e numer at io nAfter isolation of nonadherent cells, counts of NK cells (CD16,56) wereenum erated by m onoclonal antibodies (anti-CD16 and anti-CD56) con-jugated directly w ith FITC. Counts of T ce lls (CD3) were enumera ted byanti-CD3 conjugated to phycoeiythrin. (Both monoclonal antibodieswere obta ined from Becton D ickinson, Im munocytochem istry D iv is ion,Sa n Jose, Calif.). Add itional studies enum era ted LAK cell precursorsbefore stim ulation w ith IL-2 using a dual color m onoclonal antibody thatcostained for CD16,56 and CD25. The monoclona l an tibodies (20 .tl)d irectly conjugated to FITC or phycoeryth rin were added to 1 x 106 ce ll sin 20 0 1.11and incubated on ice for 30 mm before addition of 2 m l PBScontaining 2% fetal ca lf serum and 0.01% sodium azide.lmmunofluo-rescence was measured w ith a flow cytometer (FACSTAR+; BectonD ickinson , M ounta in V iew , Ca lif.). Data analysis was perform ed usingthe Consort 30 Data Management program supplied by the manufac-turer. F or every sam ple, 5000 cells were analyzed. Electronic gating ofthe lym phocyte population was performed based on forward a nd sid e-scatte r pa ram eters. T he relative proportion of each subse t w as obtainedas a percentage of the total lymphocytes counted, and the absolutenumber in each subset was calculated by m ultip ly ing the percentage ofeach subset w ith the absolute lymphocyte count derived from the whiteblood cells and differentia l count.Lymphokine-activated killer cell activityPeripheral blood m ononuclear cells are isolated as described above,resuspended at 7 x i05 cells/m i in media , and cultu red w ith or w ithout

50 units/m i of natural hum an IL-2 (Boehringer, Indianapolis, Ind.) for1 8 h at 3 7# {1 76 }Cn a 5% CO 2 incubator (19). Cyto toxicity of the stim ulatedand unstim ulated effector cells against NK resistant Daud i cell targetswas determ ined using a standard chrom ium release assay. Data fo r LAKa ctivity are p re sen tedas t he numbe r o f ly tic units in 106 cells (18) inwhich one lytic unit is de fined as the number of effector cells killing20% of the targe t cells .I nt er le uki n- 2 p roduct ionAfter density gradient separation,p erip he ra l b lo od m on on uc le ar cells( PB MC ) w er e r es us pe nd ed a t a f in al co nc en tr at io n7 X cells/rn!n10 ml of a 1:1 m ixture of RPM ! 1640 a nd D ME s up pl em en te d with 10%fetal calf serum , 4 mM glutam ine, 20 mM HEPES, 5 x 10 M 2-mer-captoe thanol (S igma, S t. Louis, Mo.), and 50 tg/ml gentamicin. Anoptima l dose of Con A (10 tg /m l; S igma) was then added and thecultures were incubated for 72 h before supernatan t harvest.

In experim ents designed to evaluate whether alterations of IL -2 pro-duction were due to PS D effects on adherent or nonadhererit cell popu-lations, adherent cells were isolated after incubation o f 1 x 1 7mononuclear cells on 150 mm diameter plastic petri d ishes fo r 1 h at37#{176}Cn 5% CO2 in air. Nonadherent cells were removed by three gentlewashes w ith warm PBS containing 5% fe ta l c alf s er um . T he a dh ere ntcells were removed by vigorous washing w ith cold PBS withou t Ca+ andMg using a 10 m l syringe fitted w ith a 22 gauge need le. V iability o fbaseline m ononuclear cells during overnight incubation before m ix ingw ith PSD-E mononuclear cells was maintained at >95% by incuba tingthe isola ted population in a 1:1 m ixture of RPM ! 1640 and DME sup-plemented w ith 10% fetal calf serum , 4 mM glutam ine, 20 mM HEPES.5 x i0 M 2-mercaptoethanol (Sigma), and 50 tg/m l gentam icin at37#{176}Cn 5% X02 in air. Nonadheren t cells and adherent cells containedin 1 x io m ononuclear cells were m ixed from the respective baselineand PSD-E conditions and resuspended at a final concentration 7 x i05cells /m l in 10 m l of a 1:1 m ixture o f supplemented RPM! 1640. Anoptima! dose of Con A (10 jig/m I; S igma) was then added and thecultures were incubated fo r 72 h before supernatant harvest.

IL-2 production by Con A-stimulated cell cultures was quantitatedu si ng t he m ur in e C TL L l in e ( 2 0) . Al th ou gh t hi s b io as sa y r es po nd s t ob ot h I L- 2 a nd I L- 4, t he a dd it io n o f a nt i- IL -2 R a nt ib od y c om pl et el yblocked the pro life rative capacity of Con A-stimulated cell cu lture su-pernatan ts (unp ublished ob servations), suggesting that IL-2 rather thanIL-4 activity is being produced and measured with the present proce-dures and CTLL clone. For each assay, a standard curve w ith knownamounts of recombinant IL-2 (250 pg/m I to 0.036 pg/m I rIL-2; Endo-

Figure 2. Effects of early-night partia l sleep depriva tion on natural k iller cell activity (mean SC M LU) in 38 healthy men. A repeated measures ANOVA demonstrated a significant time effect(F 6.3 ; df= 5,185 , P< 0.001) in which the mean va lue of NK activity a fter early-night partia ls le ep d ep riv at io n w as s ig nific an tly (P < 0.001) low er th an values afte r contro l ba seline-, baselinesleep, and recovery nights. The stippled area displays the m ean SC M o f th e th re e c on tro l b as elin evalues.


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RESULTSTable 1 show s aspects of sleep physio logy during 3nights of the partial sleep deprivation protocol: baselinesleep, PSD -E , and recovery nights. Compared w ith base-line s leep, PSD -E reduced to tal s leep time an average of3 .2 h. The tota l sleep time on the n ight o f PSD -E was


648 Vol. 10 April 1996 T he F AS EB J ou rn al IR WIN ET AL

Figure 3. Effects of early-nigh t partia l sleep depriva tion on counts of CD3 and CD16,CD56 in 32 subjects. Early sleepdeprivation had no effect on the percentage of c irculating CD3 cells (two-tailed :1.2 , df31, P0.24), but wasassociated with an overall in crease in C D3 num ber (tw o-tailed t 4 .5 , d f2 6, P< 0.001). P SD -E sig nifica ntly re du ce db ot h t he p er ce nt ag e a nd n um be r o f C D1 6, 56 ( tw o- ta il ed =7 .6 , d f3 1, P< 0.001; t3.7, df26, P< 0.001).

gen, Cambridge, Mass.) was performed to de term ine production innanograms/m illiliter. For experimental samples, cell-free c ultu re s up er -n at a nt s s e ri al lyd i lu te d ( 1 :2 t o 1 :3 2 ) w e re a d de d i n t r ip li ca t et o t heCTLL cells (4000 c el l s) .A ft e r 1 8 h the cells were pulsed w ith 0.5jiC i/well of [3H ]thym idmne (5 C i/Mm; New England Nuclear, Boston,Mass.).After 4 h, cells were harvested onto filter paper and countedu si ng a H ew le tt Packard M atrix C ounter (M eriden. C onn.). Inco rpora-tion of [3H ]thym idmne into cellu lar DNA correlates directly with theconcentration of IL-2 present in culture supernatants. The amount ofIL-2 in experimenta l wells was determ ined by compute rized fitting ofthe data on to the standard curve.

.c)-.zP..50 Basck PSD-E

F igure 4. Effects of early-night partia l sleep depriva tion on natural k illercell activ ity per number of circulating CD16,CD56 in 27 subjects. Dataillustratedrely ticunits per N K cell; loglo transfo rm ation o f these valu esdemonstrated reduced NK cellactivationaftersleep deprivation(two-t a il edt 2. 3 , d f= 2 5; P< 0.03).

S ta tis tic al a naly sisT o t es tf or t he e ff ec tso f P SD -E o n s le ep a nd t he i mm un e v ar ia bl es ,awithin-subjectsrepeated measures analysis of variance (ANOVA) wa sconducted foreach of the sleep continuityand sleep architecturevari-a bl es a nd f or the complete blood count (CBC) indices, NK activ ity , andIL-2 production.The significancevalues for repeate d m easu res A NO -V A s w er e c or re c te d f o r s p he ri c it yu si n g t he G re en h ou se - Ge is se r e ps i-! on . I m m un ol og i c a s sa y data are m is si ng f ro m s ub je ct s at s om e t im epointsfor eithertechnicalproblem s (i.e., inadequate cell recovery) orl og i st ic e a so n s ( i . e. ,C SD H um a ns Subje cts C om mitte e lim itations ont ot ala mo un t o f b lo od i n M H- CR C l ow r is k p ro to co ls .)I n t he r ep ea te dmeasures within subjectsanalyses,missing data at any time point re-sulted in the subject being excluded from that analysis.

F or coun ts of NK cells , LAK precursors, and LAK activity, whichwere assessed at b as elin e a nd a fte r PSD-E, differenceswere testedusingpaired #{163}ests. Data for NK activity (ly tic units) per number of NK cellsan d LA K a ct iv it yp er n um be r o f LA K p re cu rs or s w e re s ke we d a ndvalueswere loglotransformed tocorrectthisskewed distributionbeforeanalyses.A one-way ANOVA was used to determ ine differences in IL-2production in m ixtures of adherent (m onocyte) and nonadherent (lym -phocyte) ce lls ob tained from respe ctive b aseline and PS D condition .


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SLEE P D EP RIV AT IO N AN D IM MU NITY 64 9


about 51% of that during th e baseline sleep n igh t w ith aproportionate loss of both non-REM and REM sleep.However, re lative amounts of de lta sleep were increasedduring PSD-E at the expense of stage 2 sleep. During re-covery s leep after PSD-E, to ta l sleep time increased,whereas sleep la tency decreased compared to the base-line n ight. In add ition, s leep architecture measures re-vea led a rebound increase in the amount of de lta sleep ascom pa re d to b ase lin e.

CCBCPartia l s leep deprivation had no effect on to ta l whiteblood ce ll count w ith sim ilar numbers of circu la ting ce llsa t the two baselines, PSD -E and recovery n ights (F ig . 1).However, PSD-E produced sign ificant a ltera tions in thed is tribution of granulocytes and lymphocytes in the pe-riphera l b lood (F ig. 1) Percentage of granu locytes wassignificantly decreased after PSD -E , and both re la tive

F ig ure 5 . A) Effec tsofearl y-nig htpartia lsleep depri vatio non LAK cell activity (mean SC M L U) in 1 8 su bje cts.IL-2-stimulatedLA K ce ll ac ti v it y wass ign if icant ly ( two- ta i led t5.3, df=36, P< 0.001) lower after a n ight ofearlypa rtialsleep deprivation com pared to baseline levels. U nstim ulated cytoto xic ity against N K resistant D audiw a s n ot d if fe r en tt 1 .9 .d fr 1 7, P0.06). B) Effectsofearly-nightpartialsleep deprivationon countsof LA Kp re cu rs or sc oe xp re ss in g C D 16 ,5 6 a nd C D2 5. P SD -E h ad n o e ff ec to n e it he rt he p er ce nt ag e o r n um be r o f cellsexpressing CD16,56 and CD25 (two-tailedt0.9, df8, P0.41; t0.9, df#{176}7,#{176}O.37).) Effectsofearly -nigh tparti alslee p deprivatio n on LAK activityp er L AK precursor.Data illustratedre LAK lytic units pern umber of LA K precu rsors;loglotransf ormati onof these values d em onstrated reduced LAK cellact ivatio naftersleep deprivation(two-tailed3.O, df7, P< 0.05).


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and absolu te numbers of lymphocytes were increased bysleep deprivation. A fter recovery sleep, c ircu lating per-centages of granulocytes and lymphocytes returned tobaseline leve ls. A lthough monocyte percentages andnumbers were a ltered during the sleep deprivation proto-co l, these d ifferences were apparent on ly after the nighto f baseline sleep in the laboratory and were not due tothe effects of PSD-E.NK activityNK activity was sim ilar across the three base lines (F ig .2), and sleeping in the laboratory had no effect on va luesof cy lotoxic ity. A fte r the n igh t of PSD -E, a sign ificant(P < 0.001) reduction of NK activ ity occurred (Fig . 2) inwh ich PSD -E reduced NK activ ity to a value < 50% ofmean base line ly tic activ ity . A fte r a n ight o f recoverysleep, leve ls of NK activity re turned to va lue s c om pa rab leto those at baseline.NK cell numberTo exam ine whether the decrement o f NK activity a fte rPSD -E was due to a se lective red istribu tion of T and/orNK cells in the periphera l c ircula tion , percentages andnumbers of c ircu la ting T and NK cells were enumeratedat baseline and after PSD-E . The percentage of T ce lls(CD3) did not increase afte r PSD, a lthough number o fCD3 cells in the peripheral c ircula tion was significan tly(P < 0.001) increased afte r PSD -E as compared to base-line due to the re la tive increase in number of c ircu latinglymphocytes (F ig . 3A). For NK ce lls , bo th percentageand number of NK ce lls (CD16,56) were sign ificantly(P < 0.001) reduced after PSD-E to leve ls nearly 50%tha t found at baseline (Fig . 3B).NK activ ity per NK cellFurther ana lyses were conducted to test whether thechange of NK activ ity a fter s leep deprivation was ac-counted for by a reduction of c ircu la ting NK cells or bydecreased ly tic activity per enumerated NK cell. F irst,nonparam etric corre la tions were perform ed between val-ues of lytic activ ity and NK ce ll numbers. NK activ itywas ne ither associa ted w ith NK ce ll number a t base line(rho-O .13, P0.33) nor a fte r PSD -E (rhoO .16,P28) , and there was no re la tionsh ip between change inNK activity and change in NK ce ll number from baselineto P SD -E (rho-0 .28 , P0.15). Second, lytic activity perNK ce ll (CD16,56) was ca lcu lated: logio ly tic units perce ll w ere sign ifican tly (P < 0.05) reduced afte r PSD -E(Fig. 4). Together these findings suggest that partia ln igh t s leep depriva tion induces a reduction of NK ce llactiva tion . A decrement o f NK activity in peripheralb lood mononuclear ce lls afte r partial s leep loss does notmerely re flect a se lective red istribution of c ircu la tingpopula tions of NK cells , but ra ther ind icates an impair-ment of NK c ell a ct iv at io n.

Lymphokine-activated killer c ell a ct iv ityPSD-E induced a striking reduction of LAK cell genera-t ion. (Fig. 5A). In vitro cu lture of human periphera lblood lym phocytes w ith IL-2 results in the generation ofc yto to xic c ells . LAK ce lls lyse a broad range of in v itrotargets inc lud ing NK-sensitive and -res istant cu ltured tu-m or ce lls, and LA K activ ity assesses the action of IL-2 inprom oting diffe rentia tion, activation, and pro life ra tion ofNK cells . Induction of the LA K cells by IL-2 was exam -ined at base line and after PSD -E, and measured againstthe NK-res istant Daudi ce ll line. PSD-E resu lted in a s ig-nif icant (P < 0.01) nearly 50% suppression of IL-2stimu lated LAK activity in ly tic un its compared to base-line va lues. (F ig. 5A ) Unstim ula ted cyto toxic ity aga instthe NK resistan t Daud i cells was sim ila r be tween thePSD -E and baseline conditions (not shown).LAK activity per LAK cellTo evaluate whether the defect in LAK activ ity was dueto a d ifference in the ability o f IL-2 to generate LA K ac-tivity or is based on fewer LA K cell p recursors in the pe-riphery, the number of cells that coexpress the NK cellphenotype (CD16,56) and IL-2 receptor (CD25) wasevaluated. Neither the percentage nor number of ce lls co-express ing CD16,56 and CD25 was altered after s leeplo ss . (F ig . 5B ) Thus, when LAK activity was ca lcu latedper num ber LAK precursors, LAK cyto tox icity was aga infound to be reduced after PSD (F ig . 5C), further suggest-ing that the defect in the generation of LAK activity islike ly due to an altera tion in response of LA K precursorsto IL-2.

Additiona l s tudies enumerated ce lls that expressedCD25, but not CD16,56, and found that PS D had no ef-fect on the percentage or number of non-NK cells ex-pressing CD25 (data not shown). Consistent w ith thefindings described above on number of NK cells , th issubset o f experiments also found that partia l sleep depri-vation induced a sign ificant decline of ce lls expressingC D1 6,5 6 a lo ne.Interleukin-2 productionIL-2 , a cytokine produced by mature T helper ce lls andimportant in th e ge ne ra tio n o f cell-m ed ia te d immunity,p lays a critica l ro le in th e activa tion of NK cells . PSD -Ere su lted in s ign ifica ntly (P < 0.01) reduced IL-2 in cu l-tu res of Con A-stim ula ted periphera l b lood mononuclearcells due to e ither decreased re lease or increased utiliza-tion (F ig. 6). In contrast to the recovery of NK activityafter recovery s leep, the effects o f s leep depriva tion onIL-2 persisted after a n ight o f recovery sleep.IL-2 Production: effects of PSD on monocyte andlym phocyte cell populationsThe ro le of monocyte popula tions as compared to lympho-cytes in PSD -E reduction of IL-2 production was as-sessed by m ix ing adherent and nonadherent ce lls from


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Figure 6. Effect of early-n ight partia l s leep depriva tion IL-2 production (mean SC M ng/m l) ofC on A -s tim ula te d p erip he ra l b lood mononuc lea r cells in 17 subjects across basel ine, PSD , andrecovery n igh ts . Comparedw i th basel ine l eve ls , I L-2 produ ction w as significantlyeduced afterPSD-E and remained suppressed af ter recoverys leep (F4.7, df2,32, P< 0.02).

the respective base line and PSD-E conditions in 24 sub-jects. As shown in F ig . 7 , nonadherent (lymphocyte) ce llpopu la tions obtained after PSD -E and m ixed w ith adher-ent (m onocytes) ce lls from PSD-E condition showed asign ificant reduction of IL-2 production, consistent w iththe find ings illustra ted in F ig . 6. A sim ilar reduction ofIL-2 production was found when lym phocyte ce ll popu la-tions obta ined after PSD-E were m ixed w ith monocytesfrom the base line condition, suggesting that PSD-E in-duces an altera tion in the function of T ce lls. However, asshown in F ig . 7 , PSD -E also a lters monocyte function.W hen lymphocytes from the baseline condition werem ixed w ith monocytes from the PSD-E condition, s ignifi-cantly (P < 0.05) lower leve ls of IL-2 production werea lso found. Together, these data ind icate that PSD -E in-duces a ltera tions in both monocyte and lymphocyte popu-la tions that are associa ted w ith decrements of IL-2product ion.DISCUSSIONThese data prov ide further support for the hypothesis thatsleep has a ro le in the modulation of NK cell responsesand T cell cytokine production in humans. Loss of s leepduring the early part o f the n ight resulted in a suppres-sion of NK cell activ ity w ith decreases in both the num -bers of circu la ting NK cells and in the functiona l activityo f NK cells . The ability o f IL-2 to induce activation ofNK cells was also impaired after sleep deprivation w ithd ecre ase s in LA K activ ity a nd LA K a ctivity pe r LA K pre-cursors. Furtherm ore, m odest sleep loss induced a reduc-tion of leve ls of IL-2 in stimula ted periphera l b loodmononuclear cell p roduction that persisted at least be-yond one night o f recovery sleep. Th is defect in T ce ll re-

lease and/or u tiliza tion of IL-2 is due to the effects ofsleep loss on both the function of adherent, antigen pre-senting ce lls as well as lym phocytes.

M ultip le neuroendocrine pathways such as activation ofthe hypotha lam ic p itu itary adrena l ax is or re lease of sym -pathetic neurotransm itters have been postu la ted to m edi-ate the re la tionsh ip between sleep and immune function(21). A lthough neither Moldofsky et a !. (8) nor D ingesand co lleagues (9) found altera tions in the circad iaprhythm icity o f p lasm a cortiso l during s leep loss, an eleva-tion of sympathetic tone like ly occurs w ith s leep disrup-tion (22). Separate stud ies have shown that adrenerg icreceptor activa tion suppresses NK cytotox icity, a lters thefunction of T helper cell and/or monocytes, and sup-presses production of T ce ll cytok ines (23, 24). It is a lsopossible that s leep loss induces changes in the secretionof grow th hormone (25, 26) or mela ton in (27) which altermonocyte secretion of IL-i (28) and other ce llu lar im -mune responses (29) In addition, ACTH and cortiso l re-lease, which may or may not be e levated after partia ls leep loss(8 , 30), inh ib its IFN activation of m acrophages,expression of monocyte-derived IL-i (31), and T cell IL -2 receptor expression (32).

Though the immedia te effects of modest loss of s leepon NK cell function are robust, these a ltera tions of natu-ral and cellu lar immune function are trans ient. W ith an ight o f recovery s leep, morn ing va lues of NK activity re-turn to basa l levels . W hether th is measure of immunefunction recovers before or after the onset o f recoverysleep is not yet known. However, recovery n ight increasesof s low -wave sleep may play a cruc ial ro le in media tingthe homeosta tic recovery of immune function. Reboundincreases of s low-wave sleep typica lly occur after a n ighto f s leep deprivation-even after a night o f partial s leep


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loss (33, 34)-and slow -wave sleep has been associatedw ith increased serum concentra tions of IL-2 in humans,which in turn m ight stimula te NK cells during the recov-ery n ight (35-37). A lternative ly, endogenous IL-i is im -p lica ted in the homeosta tic recovery of slow-wave sleepafter s leep loss. Oppet at. (38, 39) have shown that theadm inis tra tion of antibod ies to IL-i antagonizes reboundincreases of slow -wave sleep in ra ts and in rabb its aftersleep deprivation. Thus, re lease of proin flammatory cy-tokines such as IL-i may occur before recovery sleep,and coord inate the induction of increases of slow -wavesleep as well as increases of NK responses and T helperc yto kin e p ro du ctio n.

The suppress ive effects of partial nig ht sle ep de priva -tion on NK cell responses and stimula ted IL-2 productioncontrast w ith the increases of NK activity, in terferon pro-duction, and IL-i-like and IL-2-like activities that havebeen reported after pro longed sleep deprivation (8-il).However, even more severe sleep loss, as has been con-ducted in ra ts , m ay resu lt in fa ilu re of immune functionw ith septicem ia and a lethal outcome (7). Together thesedata suggest that severity or duration of s leep loss is re-la ted to biphasic changes in immune responses. In viewof the im portance of cytok ines in their synerg istic regu la-tion of NK responses and in the puta tive link betweensleep and immune function, it is critica l to unambigu-ously determ ine the time course of production of cytok i-nes re la tive to s leep loss and to changes in immunee ff ec to r f un ct io n s.

Num erous considerations tem per the interpretation andgenera lizab ility o f the present find ings. To contro l for po-tentia l medica l prob lems that confound the assessment of

immune function or the eva luation of s leep physiology,the present study excluded men w ith major hea lth prob-lems or pre-exis ting s leep disorders. Thus, those w ithhealth problem s and/or ind ividuals who are already show -ing s leep disturbance such as the aged may evidencegreater decrements of NK activity or pro tracted immunedeclines than found in the present study. Furthermore,women were not included in the present study, althoughCover et a l. (40) have found that early and la te-n ight par-tia l s leep deprivation reduces NK activity in women whoare eva luated during the la te lu teal phase of the men-strual cyc le. F ina lly , the im mune variab les were assessedby a sing le b lood sample obtained at the same time ofday before and after s leep deprivation. It remains un-known whether th is effect is due to s leep per se and/or tocoinc ident changes in circad ian rhythms, i.e ., a phase de-lay re lated to the sleep loss.

The health im plica tions of a transient reduction of NK cella ctivity a nd LA K ce ll a ctivity th at fo llo ws slee p de priva tio nin hum ans are not know n. H ow ever, N K cells m edia te protec-tion against prim ary herpes virus infections. C om prom isednatura l immunity as measured in v itro by NK activity orlym phokine induction of kille r cyto tox icity m ay be of prog-nostic im portance in identifying patients at risk for recur-rence or progress ion of malignant d iseases (41-44). Inanimals, Brown et a!. (6) found that a brie f (7 h) period ofs leep deprivation can dram atically reduce host pro tectionagainst in fluenza in fection in m ice; sleep deprivation aftertertia ry intranasa l cha llenge of virus was associa ted w iths ign ifica ntly de pre sse d a nti-in flue nza an tib od y tite rs a nd th epresence of vira l partic les in lung hom ogenates. L ikew ise,E verso n (7 ) d em on stra ted th at su sta ine d sle ep de priva tio n in

Figure 7. Effects ofm ix ing adherent (monocyte)and nonadherent( ly mp ho cy te ) c ellpopulations f rom th e b as elin e a nd PSD-E cond itions on IL-2 production (m ean SC Mng/m l) in 24 sub jects. IL-2 production in the four cell m ixture groups (monocytebaseline-l ymphocy te base line; monocy te base line-l ymphocy te PSD-E; monocy tePSD-E- lymphocyte basel ine; monocytePSD-E-lymphoeytePSD-E)was signficantlydif ferent (F7.3, df= 3,69, P< 0.001). W hen compared to monocyte baseline-lym -phocyte baseline va lues, m ixtures o f monocyte base l ine- lymphocyte PSD-E, mono-cyte PSD-E- lymphocyte base li ne , and monocy te PSD-E - lymphocy te PSD-E weresignificantly (P < 0.05) lower.


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RES EARCH COMMUN IC AT IO Nrats resu lts in system ic in fection w ith at least one high lyle tha l m icrobe (i.e ., P seu do mo nas a erug ino sa, streptococ-cus) identified in the bloodstream . A dditiona l observationssuggest that sleep patterns during an in fectious d iseasem ight have prognostic va lue; Toth et a !. (45) found that afa ilu re to exh ib it increases of slow-wave sleep during Es -cherich ia coli, S trep tom yces aureus, or C andida albi cans in -fe ctio n correlated w ith in crea sed rates o f m o rta lity in rab bits .

The physio log ica l function of sleep in the maintenanceof hea lth remains unknown (21). However, these data fur-ther implica te s leep in the homeosta tic regulation ofnatura l and ce llu lar aspects of the immune system . Evena modest loss of s leep early in the n ight can lead to adecrem ent of NK activ ity , LA K ce ll activity , and lym pho-cyte production of IL-2 . E51

This work was supported by VA Merit R eview and the Nation alI ns t it ut e sf M e n ta l H ea l th ( NI M H) g ra n t (MH46867) t o M . I. Add it iona lsupport wa s provided in part b y G e ne ra l C li ni c alR es e ar ch C en te r ( MO lRR00827), t he M en ta l H e al th C li ni c al R e se ar c h C en te r ( MH 3 09 14 ) ,and the Psychopharmacology Fellowsh ip P rogram (M H 18399).

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Received for p ub lic ati on S ep te mb er 18 , 1995.Accepted fo r p ub lic at io n J an ua ry 10 . 1996.

Sleep vs. Cellular Immune

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