CLINICAL

AND

RESEARCH

REPORTS

Abnormal Diurnal Variation Cell Phenotypes and Cytotoxic John

M. Petitto, M.D., James D. Folds, Ph.D., Dana Quade, Ph.D., and Dwight

This ation

investigation

tested

of natural

and

in Circulating Natural Killer Activity in Major Depression

NK

killer

cytotoxic

the hypothesis

(NK)

activity

that

cell measures. was

depressed

The

significantly

less

Howard L. Evans,

patients

diurnal

mdc the classic clinical investigation by Sachar et al. ( I ), which showed abnormal 24-hour patterns of contisol secretion in psychotic depression, advances in the research of biological rhythms have led to new insights into the pathophysiobogy of affective illness. Distumbances in the circadian rhythms of several neurobiologicab measures-including nomadrenergic activity, contisol, melatonin, thymotmopin, temperature regulation, and the sleep-wake cycle-have been associated with major affective syndromes (2). Important components of the cellular immune system in both animals and humans also exhibit normal chronobiological pattemns of activity, and considerable evidence from basic and clinical studies has established that the CNS plays a key role in modulating various aspects of immune systern function (3). While the endogenous pacemakers that govern immunological rhythms are not yet understood, it has been posited that immune biopeniodicity may be entrained to rhythms of CNS origin (4). Natural killer (NK) cells are a heterogeneous subset of large granular lymphocytes that show cytotoxicity which is not restricted by the major histocompatibility complex. These cells are believed to be involved in the natural mesistance to tumor growth and microbial infection and in other important immunomegubatory functions, such as the Received

May

8, 1991. Microbiology, Nov.

velopment

13, 1991;

Research

of Biostatistics, School at Chapel Hill. Address Psychiatry, CB 7160, cine,

Chapel

Supported NIMH and

Resources. The authors Nancy

Dunyea,

revision

received

From the Departments the Linebergen Cancer

Center,

School

of Public reprint University

Oct.

16, 1991;

of Medicine,

694

and De-

and the Department

Health, University of North Carolina requests to Dr. Petitto, Department of of North Carolina School of Mcdi-

diurnal

of Leu-

major

Ph.D.,

depression

evidence that aspects in depression.

van-

I I NK

cells

than

in

of immune,

as

production of lymphokines (5). Several recent clinical investigations have demonstrated that circulating NK adtivity (in the morning) is abnormally low in depression (6), and to date this observation has been the most reproducible abnormality in cellular immunity associated with major depression. We recently reported (7) that low cmculating bevels of the Leu-1 I (CDI6) NK cell phenotype accompany low NK activity in depressed subjects and may be one important immunological mechanism by which NK activity is impaired in depression. Basic studies provide evidence that NK activity may be regulated by neural (e.g., noradrenergic, GABA-ergic) and endocrine (e.g., corticotnopin-neleasing factor, ACTH, cortisol, endorphin) modulators, which have been implicated in the neurobiology of depression (3). Thus, several lines of evidence suggest that abnormalities in the diurnal vanation in some mediators of CNS function associated with depression may lead to perturbations in the normal diurnab variation ofNK cell measures. To date, however, theme have been few data on such a relationship in affective disorders. To test the hypothesis that NK cell diurnal vanation is abnormal in depression, we examined circulating NK cell phenotypes and NK activity in subjects with major depression and in normal comparison subjects at both 8:00 a.rn. and 4:00 p.m. on the same day. These times were chosen because 8:00 a.m. and 4:00 p.m. have been shown to approximate the diurnal peak and nadir, mespectively,

ofNK

cell

activity

in healthy,

normal

individu-

abs (on a schedule of nocturnal rest and diurnal activity) (8). To our knowledge, this is the first study to show that the diurnal variation of a measure of cellular immunity is abnormal in patients with major depression.

Hill, NC 27599. in part by grant

thank and

by grants RR-00046

Carol Susan

MH-42625 from the

Murphy, Gray

Silva

NIH

Karen for

their

and MH-33127 from Division of Research

Tamul,

Jennifer

contributions

Blair, to

this

METHOD Forty-eight tients

research. Copyright

accepted

of Psychiatry, Medicine, Center, and the Brain and

with

M.D.,

abnormal

of levels

in 24 patients

24 normal comparison subjects. These findings provide well as neural and endocrine, chronobiology are abnormal (Am J Psychiatry 1992; 149:694-696)

S

have

variation

Ozer, M.D.

I 992

American

Psychiatric

Association.

panison

with

subjects major

subjects.

participated

depression

All subjects

Am]

in this

and

study:

24 healthy,

provided

Psychiatry

written

149:5,

24

normal

inpacorn-

informed

May

1992

CLINICAL

consent. These subjects were part of a substantially larger study of depression and immune function (7) and are the subset of subjects from whom both 8:00 a.m. and 4:00 p.m. blood samples for NK determinations were obtained on the same day. As closely as possible, the comparison subjects

were

matched

to the

patients

with

major

depres-

sion for mean age and sex distribution: mean age=30.9 years (SD=7.8) and 30.5 years (SD=8.8), respectively; percentage of men=67% and 58%. The diagnoses were assigned

by a board-certified

research

psychiatrist

(D.L.E.)

on the basis of all available clinical information and data obtained with the Schedule for Affective Disorders and Schizophrenia (SADS). The comparison subjects were mecruited by means of printed advertisement. The mean scores on the I 7-item Hamilton Rating Scale for Depnession were I 6.2 (SD=8) for the subjects with major depnession

and

subjects activity

0.8

(SD=2)

for

the

comparison

were on a schedule of nocturnal and were free of all medications

suspected

immune

effects

(e.g.,

subjects.

The

rest and diurnal with known or

psychotropics-including

antidepressants-anticonvulsants, antihistamines, antihypertensives, steroids, anti-inflammatory drugs, antibiotics) for at least 2 weeks before testing. The immune assays were performed blind to diagnosis, and all diagnoses were made blind to the results ofthe immune assays. The NK cell phenotyping and NK activity detenminations were performed with standard methods pneviousby described (7). Briefly, Leu-7 (HNK-1) and Leu1 1 (CD16) lymphocytes were identified by using Leu series

monocbonal

antibodies

and

were

counted

with

a

fluorescence-activated flow cytometer. Single-color staining was employed for the determination of the percentage of Leu-7 large granular lymphocytes (percentage of lymphocytes, 106 cells/mb), and two-color staining was used

in this

investigation

to determine

the

percentage

of Leu-1 la,c positive lymphocytes (percentage of lyrnphocytes, 106 cells/mb); the latter are the predominant population of NK active effector cells in humans. Whereas previous chronobiobogicab investigations of NK cell phenotype bevels among normal subjects have been conducted with older, less selective monocbonal antibodies (5), this is the first investigation of which we are aware that uses the anti-Leu-1 1 monocbonab antibody to assess the diurnal variation of NK cell levels. NK activity determinations were performed for a subset of patients ( 1 8 depressed patients, 1 1 comparison subjects) with chromium-labeled, NK-sensitive K562 tumom cells as the target. The percentage of specific lysis of tumor targets at each of five effectom/target cell ratios ( I 2.5 to 200: 1 ), the area under the concentration-response curve, and the mean NK activity (the average of the five effectom/tanget cell ratios) were calculated for each

AND

RESEARCH

REPORTS

between the patients with major depression and the normal comparison subjects, and Pearson partial correlations were performed to examine the relationship between Hamilton severity mating and diurnal vanation for each immune measure. For statistical analyses, immune values were log-transformed to normalize group variance. Adjusted logged values were unbogged for presentation in the text. The mean and standard deviation are reported, and two-tailed tests were used throughout. The group differences in diurnal variation of the dependent immune variables reported are cornputed difference scones analyzed by ANCOVA.

RESULTS Statistical analyses of the computed difference scones demonstrated that the subject group with major depression had significantly less diurnal variation of Leu-1 1 NK cell levels (+1%) than the group of normal comparison subjects (-14%) between 8:00 a.rn. and 4:00 p.m. (F= 3.94, df=1, 44, p=O.OS). The mean level of Leu-1 1 cells (percentage of lymphocytes, 106 cells/mI) decreased from 12.6 (SD=S.9) to 11.0 (SD=4.9) between 8:00 a.m. and 4:00 p.m. for the normal comparison subjects. For the patients with major depression the mean Leu-1 1 levels were 10.6 (SD=S.9) at 8:00 a.m. and 10.8 (SD=S.4) at 4:00 p.m. The diurnal variation in mean NK cytotoxic activity was also found to be significantly less for the subjects with major depression (-24%) than for the normal comparison subjects (-45%) (F=6.72, df=1, 25, p=O.O2). In addition, these differences were significant for each of the five effector!tamget cell ratios tested-200:1 (F=S.99, df=1, 2S,p=O.O2), 100:1 (F=4.94,df=1, 2S,p=O.O4),SO:l (F=S.71, df=1, 25, p=O.O2), 25:1 (F=4.47, df=1, 25, p= 0.04), and 12.5:1 (F=S.64, df=1, 25, p=0.03)-and for the NK area under the curve (F=6.67, df=1, 25, p=0.O2). The mean NK activity decreased from 44 (SD=13) to 24 (SD=12) between 8:00 a.m. and 4:00 p.m. for the normal comparison subjects and from 38 (SD=13) to 29 (SD=8) for the subjects with major depression. The levels of Leu-7 cells did not differ significantly between the subject groups (F=2.S8, df=1, 43, p= 0.12). Partial correlations examining the relationship between Hamilton depression score and diurnal vanation

in the

three

immune

cant. There were no significant den differences in the diurnal

measures

were

not

depression-related variation of these

signifi-

genimmune

measures.

DISCUSSION

subject.

For the individual dependent immune variables cxamined, a difference score was computed for each subject (4:00 p.m. value minus 8:00 a.m. value). Any potential variance in the dependent immune variables due to age or gender was controlled for in all statistical analyses.

ing

for

Am

]

Analysis

age

and

Psychiatry

of

gender,

I 49:5,

covaniance

was

May

(ANCOVA),

used

1992

to test

covary-

for differences

The results reported here indicate that the diurnal variation in NK activity is abnormal in patients with the syndrome of major depression. Normal circadian vanation in NK cytotoxic activity varies as a function of changes in both the bevel of peripheral blood NK cells and the activation state of those cells oven time (8). The Leu-1 I antigen marker is present on virtually all cells in

695

CLINICAL

AND

RESEARCH

REPORTS

the peripheral blood that are known to have functionally meaningful NK activity, and peripheral blood LeuI I NK cell levels are positively correlated with NK adtivity in normal subjects (5). As expected, the normal comparison subjects in this study exhibited substantial diurnal variation in circulating Leu-11 NK cell levels that tracked the diurnal variation of NK activity. In contrast, the patients with major depression had significantly

less

diurnal

change

in NK

activity

and

almost

no

variation in Leu-1 1 NK cell bevels. Peripheral blood NK cell phenotype bevels are thought to reflect lymphocyte migration or recirculation between immune organs (e.g., spleen) and blood. In healthy subjects the response of peripheral blood NK cells to stimulatory and inhibitory immunomegulatomy signals appears to differentially vary during the circadian

cycle

(8).

Hence,

our

finding

that

there

is greaten

diurnal variation in NK activity than in Leu-1 1 NK cell levels among normal subjects is in keeping with cvidence in the immunological literature which suggests that the daily biopeniodicity of NK activity is a function of changes in both blood levels of NK cells and the adtivation state of those cells. Given the finding that diurnab variation between 8:00 a.m. and 4:00 p.m. for both Leu-1 1 NK cell levels and NK activity is abnormal in patients with major depression, it will be important in future studies to examine multiple time points (e.g., every 3 hours) so that time series analyses can be used to systematically assess chronobiobogical differences (e.g., phase differences) throughout the full circadian cycle. Moreover, determining the dynamic temporal melationship between NK measures and neumomodulators that have been suggested to play a mole in NK cell megulation (e.g., corticotropin-releasing factor, cortisol, endomphin) will be important in testing hypotheses about their role in NK cell biopeniodicity. Diurnal variation in Leu-7 lymphocyte levels did not differ significantly between the groups in our study, possibly

because

the

peniodicities

of subgroups

and

indicates bograft

696

other

that rejection,

areas

of medicine.

susceptibility and tumor

Experimental

asymptornatic

evidence

to microbial infection, algrowth, as well as immuno-

HIV-infected

individuals

may

manifest

disruptions of the normal cincadian cycle of CD4 (T helper) cells and that such disruptions may predate both symptoms of HIV-spectrum disease and, in some cases, reductions in CD4 cell counts. Since HIV infection affects the CNS as well as the immune system, it is possible that abnormal CD4 cincadian rhythms may be secondany to chronobiobogical changes in the CNS. In conclusion, by demonstrating that the normal diurnab variations in both circulating levels of the Leu-1 1 NK cells and NK activity are disrupted in patients with major depression, these data provide the first evidence that measures of immunological, as well as neural and endocrine, diurnal variation are abnormal in depression. Systematic investigation of interactions between complex neural, endocnine, and immune chronobiological rhythms may bead to greaten basic understanding of the biology of brain-immune interactions in depression and perhaps other neuropsychiatric syndromes.

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Gatti G, Rossana C, Santoni ML, Canignola R, Masera R, Delponte D, Salvadoni A, Angeli A: Circadian variations of interferon-induced enhancement of human natural killer (NK) cell activity. Cancer Detect Prey 1988; 12:431-438 Reinbeng A, Smolensky MH (eds): Biological Rhythms and Mcdicine: Cellular, Metabolic, Physiopathologic and Pharmacologic

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Morley JE, Kay NE, Solomon GF, Plotnikoff NP: Neuropeptides: conductors of the immune orchestra. Life Sci I 987; 41:527-544 Maestroni GJM, Conti A, Pienpaoli W: Melatonin, stress and

immune

of Leu-7

positive cells are affected differently in major depression. Unlike the lymphocytes of the Leu-1 I NK cell phenotype, Leu-7 antigens are found on a variable percentage (approximately 50%) of functionally active NK cells, as well as on subsets of T lymphocytes, some of which have patterns of rhythmic variation that differ from that of NK cells. The clinical implications of biological rhythms have been of great interest to clinical researchers in psychiatry

pharmacologic responses, among other clinically mdcvant end points, vary significantly throughout the circadian cycle (9). One clinical study (10) indicated that

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Psychiatry

I 49:5,

Ma)’

1992