Circulating Natural Killer Cell Phenotypes in Men and Women With Major Depression Relation to
Cytotoxic Activity and Severity of Depression
Dwight L. Evans, MD; James D. Folds, PhD; John M. Petitto, MD; Robert N. Golden, MD; Cort A. Pedersen, MD; Mark Corrigan, MD; John H. Gilmore, MD; Susan G. Silva, MS; Dana Quade, PhD; Howard Ozer, MD, PhD effects of major depression on peripheral blood natural killer cell phenotypes and natural killer cell activity were studied by comparing depressed and normal control subjects. Depressed subjects exhibited (1) significant reductions in Leu-11 (CD16) natural killer effector cells and natural killer cell activity and (2) a dissociation of the normal positive correlation between the percentage of Leu-11 cells and natural killer cell activity. These findings suggest that alterations in the availability and the killing capacity of circulating Leu-11 natural killer cells appear to be responsible for depression-related reductions in natural killer cell activity. Moreover, men with major depression showed marked reductions in Leu-11 cells, natural killer cell activity, and Leu-7 (HNK-1) lymphocytes compared with normal control men. By contrast, depressed women did not differ significantly from normal control women on any of these three immune function measures. Severity of depression as assessed by Hamilton Rating Scale for Depression scores was not associated with natural killer cell activity or Leu-7 lymphocyte levels in either men or women with major depression. Hamilton Rating Scale for Depression severity ratings were, however, strongly inversely correlated with Leu-11 lymphocyte counts among men, but not women, with major depression. These data begin to elucidate the immunological mechanisms by which natural killer cell activity is altered in depression and suggest that some measures of immunity may be differentially affected in male and female subjects with the syndrome of major \s=b\ The
depression.
(Arch Gen Psychiatry. 1992;49:388-395)
the 1977 report by Bartrop and colleagues1 dem¬ reduced lymphoproliferative responses to mitogens in the recently bereaved, extensive work has been done investigating cellular immune function in stress and depression. To date, however, considerable
Sinceonstrating Accepted
for publication April 25, 1991. Departments of Psychiatry (Drs Evans, Petitto, Golden, Pedersen, Corrigan, and Gilmore and Ms Silva), Microbiology (Dr From the
Folds), and Medicine (Drs Evans and Ozer), the General Clinical Research Center (Dr Golden), and the Lineberger Cancer Center (Dr Ozer), School of Medicine; and the Department of Biostatistics, School of Public Health (Dr Quade), University of North Carolina,
Chapel
Hill. Presented in part at the American Psychiatric Association Annual Meeting, New Orleans, La, May 14,1990; and the American College of Neuropsychopharmacology Annual Meeting, San Juan, Puerto Rico, December 10, 1990. Reprint requests to Department of Psychiatry, University of Florida, PO Box 100256, JHMHSC, Gainesville, FL 36210 (Dr Evans).
lack of agreement exists as to what specific immune alter¬ ations are associated with major depression. In particular, some studies have found reduced lymphoproliferative responses to mitogens in patients with major depression,26 while other studies have found no such relationship.7"9 Recent data have shown that severity of depression and age may be two important variables asso¬ ciated with depression-related immune alterations,9 thus suggesting that negative studies may reflect findings from less severely depressed and/or younger patients. Early studies of depression and immunity focused on lymphocyte response to mitogens as a measure of cellu¬ lar immunity. Recent studies have begun to examine im¬ mune factors that are thought to reflect more functionally significant and specific measures of cellular immunity, such as natural killer (NK) cell activity (NKA). Natural killer cells are a heterogeneous subset of large granular lymphocytes that show cytotoxicity that is not restricted the major histocompatibility complex. These cells pos¬ sess a variety of functions, such as natural resistance against tumor growth and microbial infections, as well as the production of lymphokines.1011 Although the largest study to date did not find changes in NKA in depressed patients,9 several recent investiga¬ tions have demonstrated that NKA is reduced in de¬ pressed patients.6-1216 Few empirical data are available, however, regarding the mechanism(s) by which these re¬ ductions in NKA occur. Alterations in NKA can be pro¬ duced by two principal mechanisms: decreased availabil¬ ity of NK cells in peripheral circulation, or perturbations in the killing "capacity" of NK effector cells.1117"19 Basic studies have found that NKA is sensitive to many factors, including classic immunomodulators (eg, interleukin 2, interferons), as well as neural (eg, catecholamines) and neuroendocrine (eg, corticotropin-releasing factor, corti¬ cotropin, ß-endorphin) modulators that have been impli¬ cated in the neurobiology of depression.20"22 To under¬ stand how depression-related changes in specific neural and endocrine peptide systems may regulate NK cell function in this syndrome, it is essential to determine how key NK cell populations responsible for NKA are altered in depression. Therefore, the simultaneous measurement of circulating NK cell populations to¬ gether with NKA determinations should provide vital information that will begin to elucidate the immune mechanisms by which NKA is altered in this condition. Such information will be important for directing future studies designed to examine how neural and endocrine changes associated with depression may mediate this function of cellular immunity.
by
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Characteristic
Sex,
Normal Controls (n = 48)
F/M
Depressed =
19/29
Age,
y Mean±SD
Range HAM-D scores, mean±SD*
Patients
(n 44) 16/28
28±1.0
31 ±1.2
(18.9-45.6)
(18.6-46.6)
0.8±0.3
17.3±1.1
•Hamilton Rating Scale for Depression (HAM-D) scores were ob¬ tained on 35 normal control subjects and 35 depressed patients.
Natural killer cells can be identified in the peripheral blood using specific fluorescent monoclonal antibodies. The Leu-7 (HNK-1) antigen marker is known to identify large granular lymphocytes, some of which express NK activity,23"25 and the Leu-11 (CD16) marker is present on virtually all cells in the peripheral blood that are known to have functional NK activity.J1'26-27 We hypothesized that (1) Leu-11 cell populations in the peripheral blood would be lower in patients with major depression compared with normal control subjects, (2) reductions in the Leu-11 cell populations in the peripheral blood of depressed pa¬ tients would be responsible for reductions in NKA, and (3) severity of depression912 rather than the presence or absence of depression would be correlated with these changes in the Leu-11 NK cell population and NKA. To test these predictions, we assessed both quantitative (NK cell phenotypes) and qualitative (NK activity) NK mea¬ surements in depressed patients compared with normal control subjects. To our knowledge, this is the first study to investigate both levels of circulating NK cells and NKA
in
major depression.
SUBJECTS AND METHODS
Subjects
study was approved by the University of North Carolina (Chapel Hill) School of Medicine Committee for the Protection of the Rights of Human Subjects, and all subjects provided writ¬ ten informed consent. We examined 92 subjects: 44 patients with major depression (28 men and 16 women) and 48 healthy con¬ trol subjects (29 men and 19 women; Table). Healthy control subjects and depressed patients were matched for mean age and sex as closely as possible. The age range for the depressed group was 18.6 to 46.6 years (mean±SEM, 30.8±1.2 years), and for the normal controls it was 18.9 to 45.6 years (28.2±1.0 years). All depressed subjects were studied through the University of North Carolina Mental Health Clinical Research Center (MHCRC). To include a broad range of depressed subjects, we studied subjects hospitalized in the MHCRC units at the University of North Carolina Neuropsychiatrie Hospital and the Dorothea Dix Hospital, Raleigh, NC, as well as four psychiatric outpatients examined on the University of North Carolina Gen¬ eral Clinical Research Unit. All depressed subjects met DSMIII-R criteria for major depression. Diagnoses were assigned by a board-certified research psychiatrist (D.L.E.), based on all available clinical data, including chart review of presenting signs and symptoms, psychiatric history, past treatment response, medical history, laboratory data, family history, and daily progress notes, as well as data generated from the Schedule for This
Affective Disorders and Schizophrenia-Lifetime version. Labo¬ ratory studies were performed in a double-blind fashion: diag¬ noses were assigned "blind" to all immune data, and laboratory data were obtained on coded blood samples. Depression ratings were obtained within 3 days of immune testing and consisted of
the 17-item Hamilton Rating Scale for Depression (HAM-D),28 which was administered by a psychiatric research nurse or fel¬ low of the MHCRC; the intraclass coefficient of correlation for these raters is R .91. The mean ± SEM depression ratings of the subjects with major depression and the normal control subjects are given in the Table. Healthy, normal control subjects were recruited by printed ad¬ vertisement and admitted to the University of North Carolina General Clinical Research Unit. The control subjects underwent the same systematic medical and psychiatric assessment as the depressed subjects, including physical examination and medical history, laboratory screening (complete blood cell count, serum electrolyte, urea nitrogen, creatinine, calcium, and protein val¬ ues, liver function tests, and thyroid function tests), the Schedule for Affective Disorders and Schizophrenia-Lifetime version, and the depression ratings. Control subjects were free of psychiatric illness, and all depressed and control subjects were free of any acute or chronic medical conditions known to affect immune function and were free, for at least 2 weeks before testing, of sub¬ stances or medications known to influence immune function. =
Procedures
Subjects took nothing by mouth starting at midnight before the drawing of blood the next morning. An intravenous line was
started at 8 am in an antecubital vein and maintained patent with slow drip of normal saline solution. To control for circadian ef¬ fects, peripheral blood for the immune measurements was ob¬ tained at 9:10 am. We obtained NK cell phenotype measures on all subjects to address hypothesis 1. To address hypothesis 2, NKA measurements were obtained on a subset of our sample (30 patients and 19 controls). The immune assays were performed in the Psychoneuroimmune Laboratory of the MHCRC. To reduce assay variance, fresh internal control samples were obtained from normal healthy controls and assayed for the monoclonal antibody determinations of NK cell populations; for the NK cell activity assays, internal control samples were cryopreserved following lymphocytapheresis.29 Ficoll-purified washed mononuclear cell populations were divided in aliquote and cryopreserved in the vapor phase of a liquid nitrogen freezer and thawed for each as¬ say. Each functional assay thus compared experimental subjects (either normal controls or depressed patients) with standardized frozen control samples. Monoclonal Antibody and Flow Cytometry Techniques.— Peripheral blood samples were collected into heparinized vac¬ uum (Vacutainer) tubes. The Leu series of monoclonal antibod¬ ies (Becton Dickinson, Mountain View, Calif) was used to measure the predominant reactivity as described previously.30 a
Briefly, mononuclear cells were partially purified by centrifugation through lymphocyte separation medium (Bionetics Labora¬ tory Products, Rockville, Md). Lymphocytes were collected by centrifugation and washed twice in RPMI1640 and adjusted to 1 107/mL. The cells were centrifuged in a centrifuge (SerofugeClay Adams Ine, Parsippany, NJ) and washed again in cold water-bovine serum albumin-azide-phosphate-buffered saline (pH 7.2). After the last wash step, the supernatant was poured off and 5 µ of the fluorescein-labeled monoclonal antibody re¬
was added for detection of cell surface markers. AntiLeu-7 and anti-Leu-HA monoclonal antibodies were used for their predominant reactivity with cells of the NK phenotype. A background control was used for nonspecific staining with fluorescein-labeled anti-IgGl. The cells were incubated with the monoclonal antibodies at 40°C for 45 minutes. Stained cells were washed twice with 1 mL of cold water-bovine serum albuminazide-phosphate-buffered saline, fixed by the addition of 1% paraformaldehyde, and stored in the dark at 4°C until examina¬ tion with a flow cytometer (FACScan, Becton Dickinson). Refer¬ ence ranges were determined by evaluation of normal peripheral blood controls. NKA Assay.—Natural killer cell cytotoxicity was measured in vitro using a chromium release assay described in detail else-
agent
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Fig 1.—Analysis ofcovariance adjusted forage revealed a significant difference between controls and patients with major depression (F[T, 731 7.46, P 008). Leu- values were logged for analysis and transferred back to original units (mean±SEM) for clarity of presen¬ tation. =
=
.
where.31 Briefly, following two washes in RPMI 1640 with 10% fetal bovine serum, varying dilutions of thawed effector cells from each subject (and internal control sample cells) were added (100 µ ) to triplicate U-bottom microtiter wells (Costar, Cam¬ bridge, Mass). Target cells were the NK-sensitive K562 tumor cell line labeled with 200 µ of sodium chromate-51 (10.8 MBq/mg; New England Nuclear, Boston, Mass) for 1 hour at 37°C. Following the labeling step, target cells were then washed three times in the above medium and counted, and a 100-µ aliquot (5000 cells) was added to wells containing effector cells. The mi¬ crotiter plates were then centrifuged for 5 minutes at 800 rpm and incubated for 18 hours in a humidified 95% air, 5% carbon diox¬ ide atmosphere. Following incubation, the plates were recentrifuged at 1200 rpm for 10 minutes. The supernatant from each well was collected and counted in a gammagraphic counter (LKB Co, Turku, Finland). Cytotoxicity was measured across five effectontarget cell (E:T) concentrations (200:1 to 12.5:1) for each subject. The percentage of lysis was determined using the
following equation: %Lysis (exp cpm-spont cpm)/(max cpm spont cpm) 100, where exp cpm was defined as the counts released into the medium by target cells when assayed =
-
with effector cells; max cpm was defined as known counts added in 5000 target cells; and spont cpm was defined as the counts re¬ leased by target cells in the presence of medium alone. Sponta¬ neous release was less than 5% of maximum release in all assays.
Statistical
Analysis
The Leu-7 and Leu-11 raw values were log transformed to sta¬ bilize group variance, and these logged values were then used as dependent measures in the statistical analyses. Analysis of covariance (ANCOVA) procedures, controlling for age effects,
Fig 2.—Analysis of covariance adjusted forage revealed a significant between controls (solid bars) and patients with major .015) but not in depression (open bars) in male (F[1,431 6.44, female subjects (FIT,271 =¡.57, P .22). Leu-H values were logged for analysis and transferred back to original units (mean±SEM) for clarity of presentation. difference
=
=
=
performed to examine the group differences between the patients with major depression and normal controls, as well as depression-control group differences for men and women sep¬ arately. The ANCOVAs for repeated measures were used to ex¬ amine group differences on NKA, where cytotoxicity was mea¬ sured at five E:T ratios. Correlational procedures were used to assess the relationship between severity of depression and the various immune measures, as well as the relationship between were
NK
phenotype and NKA.
All statistical tests
were
two-tailed.
RESULTS Pearson correlation analyses indicated that there was a signif¬ icant positive relationship between age and percentage of Leu-7 lymphocytes for all subjects in the study (r=.27, =.01). This result is consistent with other studies in the immunological lit¬ erature, showing that there is a positive correlation between age and percentage of Leu-7 lymphocytes from birth to senes¬ cence.11·32 In all subsequent analyses, age was used as a covariate to control for the effects of age on each immune measure and for consistency among data analyses. For clarity of presentation, the values presented in the figures for Leu-7 and Leu-11 are un¬
tagged values adjusted for age.
NK Cell Phenotypes in Peripheral Blood Patients with major depression were 27.4% lower in percent¬ age of Leu-11 lymphocytes compared with normal controls (F[l,73] 7.46, P=.008; Fig 1). As depicted in Fig 2, men with major depression were 31.6% lower in their percentage of Leu-11 lymphocytes when compared with male normal controls women with major depres¬ (F[l,43] 6.44, .015). sion also had a lower percentage of Leu-11 lymphocytes than fe=
=
=
Although
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Fig 3.—Analysis of covariance adjusted for age revealed a trend to¬ ward lower percentage of Leu-7 lymphocytes in patients with major depression compared with controls; however, statistical signifi¬ cance was not reached (F[¡, 861 3.62, .06). Leu-7 values were logged for analysis and transferred back to original units (mean±SEM) for clarity of presentation. =
male controls, this difference
was
=
not
(F[l,27] 1.57, P=.22).
Peripheral blood Leu-7 cell counts were lower (26.7%) in the group with major depression; however, this difference did not reach statistical significance (F[l,86] 3.62, .06; Fig 3). Figure 4 illustrates that men with major depression had 40% lower Leu-7 lymphocyte levels than male controls (F[l,53] 5.81, P= .02), but there was no difference between the women with major depression and female controls (F[l,30] 0.17, P=.68). =
=
=
Natural Killer Cell
difference between controls (solid bars) and patients with ma¬ .02) but not in jor depression (open bars) in male (F[¡,531 5.8¡, female subjects (F[¡, 30] 0.¡7, P .68). Leu-7 values were logged for =
=
analysis and transferred back clarity of presentation.
Activity
Natural killer cell activity data were available on a subset of the subjects in the lymphocyte phenotype analysis; there was no significant difference in the demographic and clinical character¬ istics of these subjects compared with the subjects in the pheno¬ type analysis. There was a graded lysis of K562 tumor target cells by effector cells of subjects from both diagnostic groups over the five E:T ratios tested, 200:1 to 12.5:1. A repeated-measures ANCOVA revealed a significant main effect of diagnosis (F[l,46] 6.66, =.01). Planned comparison of means analysis showed that subjects with major depression had lower NKA than control subjects at each E:T ratio, ranging from 25.1% lower at 200:1 (P= .02) to 46.7% lower at 12.5:1 (P= .03; Fig 5). As de¬ picted in Fig 6, when the major depression and control groups were divided by gender, men with major depression exhibited significantly lower NKA than male controls both when the data were collapsed across all of the E:T ratios (F[l,24] 7.82, P= .01) and at each of the five E:T ratios (31.7% to 51.9%; P= .006 to .02). Conversely, women with major depression had lower NKA but did not differ significantly from female controls at any of the five E:T ratios (5.0% to 33.6%; P= .38 to .82). =
=
=
=
to
original
values (mean±SEM) for
NK Cell Measures and HAM-D Scores
statistically significant
=
=
Fig 4.—Analysis of covariance adjusted for age indicated a signifi¬
cant
No significant relationship was noted between HAM-D sever¬ ity rating and either mean NKA or percentage of Leu-7 lympho¬ cytes in the depressed group. However, when the potential ef¬ fects of age and gender were controlled, the Pearson partial cor¬ relation between HAM-D rating and percentage of Leu-11 lymphocytes approached statistical significance (r=-.33, P= .06). Further analysis of the data by gender revealed a highly significant negative correlation between HAM-D rating and per¬ centage of Leu-11 lymphocytes in men with major depression (r= —.71, P= .0005), but not in women with major depression (r=.12, =.69). Natural killer cell activity was not related to HAM-D ratings in either men (r= .20, P= .49) or women (r= .15,
=.66).
Relationship of Leu-11
NK Cell
Phenotype to NKA
partial correlation (subtracting the variance ac¬ counted for by age and gender) confirmed the presence of the expected relationship between percentage of Leu-11 lympho¬ cytes and NKA in normal control subjects (Fig 7;r= .58, P= .03). Conversely, as seen in Fig 7, there was a negative relationship A Pearson
between percentage of Leu-11 cells and mean NKA among pa¬ tients with major depression (r= .36, P= .08). —
COMMENT These data demonstrate that NK effector cell popula¬ tions are reduced in depression, which confirms the find¬ ings from our earlier pilot study.30 Consistent with our prediction, significant reductions in Leu-11 lymphocyte counts were found in the group with major depression,
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200:1
100:1
25:1
50:1
12.5:1
EffectorTarget Cell Ratio of covariance adjusted for age showed significant differences (mean±SEM) in natural killer cell (NK) percent lysis for men at each effector.target cell ratio (F]¡,241 7.82, P .07): 200:¡ ( .006), ¡00:¡ ( .02), 50:¡ ( =.04), 25:1 ( .07), and ¡2.5:1 ( .07). This difference was not evidenced in women (FU, ¡91 0.30, P .59):200:¡ (P .83), ¡00:1 (P .73), 50:1 (P .38), 25:1 (P .65), and 12.5:1 (P .49). Solid triangles indicate male con¬ trols (n 70); solid circles, female controls (n 9); open triangles, men with major depression (n 77); and open circles, women with major depression (n 13).
Fig 6.—Analysis
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
lyse target cells. Killing capacity can be altered in sev¬ eral ways, including dysfunctional target recognition, impaired release of cytotoxic factors, decreased sensitiv¬ ity to regulatory cell signals, or altered regulatory cell function. Our results demonstrate that circulating NK cells labeled by Leu-11 monoclonal antibodies are reduced in depression, consistent with our prediction that de¬ creased Leu-11 cells would account for reductions in NKA. However, the expected positive correlation be¬ tween Leu-11 lymphocyte levels and NKA was highly significant in our normal control subjects18-25 but absent (and in fact negative) among depressed subjects. There¬ fore, reduced NKA in the depression group cannot be ex¬ plained solely on the basis of decreased circulating Leu-11 NK cells in the peripheral blood. These data suggest that alterations in both circulating levels of NK cells and their killing capacity are responsible for depression-associated reductions in NKA. When the data were analyzed by gender, there were striking depression-related gender differences for each of the three immune measures examined in this study. Male patients with major depression exhibited significant re¬ ductions in Leu-11 cell counts, NKA, and Leu-7 lympho¬ cyte counts compared with male controls. Conversely, women with major depression were not significantly dif¬ ferent from female controls for any of the three dependent variables studied. Thus, our hypothesis that Leu-11 lym¬ phocytes and NKA would be altered in depression was confirmed in men. We did not confirm this hypothesis in women. Our findings in women may be due to a weaker effect in women, a smaller female sample size, or possi¬ bly a floor effect in women. No study to date has explic¬ itly assessed whether major depression differentially af¬ fects immune function in male and female subjects. There is also a lack of preclinical and clinical data examining whether immunological responses to stress differ among male and female subjects. No previous study has assessed gender differences in NK cell phenotyping in major or
Fig 5.—Analysis of covariance adjusted for age showed that 30 sub¬ jects with major depression (open bars) had lower natural killer cell (NK) percent lysis (mean±SEM) than 79 controls (solid bars; Fit, 46] 6.6, P
Cytotoxic Activity and Severity of Depression
Dwight L. Evans, MD; James D. Folds, PhD; John M. Petitto, MD; Robert N. Golden, MD; Cort A. Pedersen, MD; Mark Corrigan, MD; John H. Gilmore, MD; Susan G. Silva, MS; Dana Quade, PhD; Howard Ozer, MD, PhD effects of major depression on peripheral blood natural killer cell phenotypes and natural killer cell activity were studied by comparing depressed and normal control subjects. Depressed subjects exhibited (1) significant reductions in Leu-11 (CD16) natural killer effector cells and natural killer cell activity and (2) a dissociation of the normal positive correlation between the percentage of Leu-11 cells and natural killer cell activity. These findings suggest that alterations in the availability and the killing capacity of circulating Leu-11 natural killer cells appear to be responsible for depression-related reductions in natural killer cell activity. Moreover, men with major depression showed marked reductions in Leu-11 cells, natural killer cell activity, and Leu-7 (HNK-1) lymphocytes compared with normal control men. By contrast, depressed women did not differ significantly from normal control women on any of these three immune function measures. Severity of depression as assessed by Hamilton Rating Scale for Depression scores was not associated with natural killer cell activity or Leu-7 lymphocyte levels in either men or women with major depression. Hamilton Rating Scale for Depression severity ratings were, however, strongly inversely correlated with Leu-11 lymphocyte counts among men, but not women, with major depression. These data begin to elucidate the immunological mechanisms by which natural killer cell activity is altered in depression and suggest that some measures of immunity may be differentially affected in male and female subjects with the syndrome of major \s=b\ The
depression.
(Arch Gen Psychiatry. 1992;49:388-395)
the 1977 report by Bartrop and colleagues1 dem¬ reduced lymphoproliferative responses to mitogens in the recently bereaved, extensive work has been done investigating cellular immune function in stress and depression. To date, however, considerable
Sinceonstrating Accepted
for publication April 25, 1991. Departments of Psychiatry (Drs Evans, Petitto, Golden, Pedersen, Corrigan, and Gilmore and Ms Silva), Microbiology (Dr From the
Folds), and Medicine (Drs Evans and Ozer), the General Clinical Research Center (Dr Golden), and the Lineberger Cancer Center (Dr Ozer), School of Medicine; and the Department of Biostatistics, School of Public Health (Dr Quade), University of North Carolina,
Chapel
Hill. Presented in part at the American Psychiatric Association Annual Meeting, New Orleans, La, May 14,1990; and the American College of Neuropsychopharmacology Annual Meeting, San Juan, Puerto Rico, December 10, 1990. Reprint requests to Department of Psychiatry, University of Florida, PO Box 100256, JHMHSC, Gainesville, FL 36210 (Dr Evans).
lack of agreement exists as to what specific immune alter¬ ations are associated with major depression. In particular, some studies have found reduced lymphoproliferative responses to mitogens in patients with major depression,26 while other studies have found no such relationship.7"9 Recent data have shown that severity of depression and age may be two important variables asso¬ ciated with depression-related immune alterations,9 thus suggesting that negative studies may reflect findings from less severely depressed and/or younger patients. Early studies of depression and immunity focused on lymphocyte response to mitogens as a measure of cellu¬ lar immunity. Recent studies have begun to examine im¬ mune factors that are thought to reflect more functionally significant and specific measures of cellular immunity, such as natural killer (NK) cell activity (NKA). Natural killer cells are a heterogeneous subset of large granular lymphocytes that show cytotoxicity that is not restricted the major histocompatibility complex. These cells pos¬ sess a variety of functions, such as natural resistance against tumor growth and microbial infections, as well as the production of lymphokines.1011 Although the largest study to date did not find changes in NKA in depressed patients,9 several recent investiga¬ tions have demonstrated that NKA is reduced in de¬ pressed patients.6-1216 Few empirical data are available, however, regarding the mechanism(s) by which these re¬ ductions in NKA occur. Alterations in NKA can be pro¬ duced by two principal mechanisms: decreased availabil¬ ity of NK cells in peripheral circulation, or perturbations in the killing "capacity" of NK effector cells.1117"19 Basic studies have found that NKA is sensitive to many factors, including classic immunomodulators (eg, interleukin 2, interferons), as well as neural (eg, catecholamines) and neuroendocrine (eg, corticotropin-releasing factor, corti¬ cotropin, ß-endorphin) modulators that have been impli¬ cated in the neurobiology of depression.20"22 To under¬ stand how depression-related changes in specific neural and endocrine peptide systems may regulate NK cell function in this syndrome, it is essential to determine how key NK cell populations responsible for NKA are altered in depression. Therefore, the simultaneous measurement of circulating NK cell populations to¬ gether with NKA determinations should provide vital information that will begin to elucidate the immune mechanisms by which NKA is altered in this condition. Such information will be important for directing future studies designed to examine how neural and endocrine changes associated with depression may mediate this function of cellular immunity.
by
Downloaded From: http://archpsyc.jamanetwork.com/ by a University of California - San Diego User on 12/15/2016
Characteristic
Sex,
Normal Controls (n = 48)
F/M
Depressed =
19/29
Age,
y Mean±SD
Range HAM-D scores, mean±SD*
Patients
(n 44) 16/28
28±1.0
31 ±1.2
(18.9-45.6)
(18.6-46.6)
0.8±0.3
17.3±1.1
•Hamilton Rating Scale for Depression (HAM-D) scores were ob¬ tained on 35 normal control subjects and 35 depressed patients.
Natural killer cells can be identified in the peripheral blood using specific fluorescent monoclonal antibodies. The Leu-7 (HNK-1) antigen marker is known to identify large granular lymphocytes, some of which express NK activity,23"25 and the Leu-11 (CD16) marker is present on virtually all cells in the peripheral blood that are known to have functional NK activity.J1'26-27 We hypothesized that (1) Leu-11 cell populations in the peripheral blood would be lower in patients with major depression compared with normal control subjects, (2) reductions in the Leu-11 cell populations in the peripheral blood of depressed pa¬ tients would be responsible for reductions in NKA, and (3) severity of depression912 rather than the presence or absence of depression would be correlated with these changes in the Leu-11 NK cell population and NKA. To test these predictions, we assessed both quantitative (NK cell phenotypes) and qualitative (NK activity) NK mea¬ surements in depressed patients compared with normal control subjects. To our knowledge, this is the first study to investigate both levels of circulating NK cells and NKA
in
major depression.
SUBJECTS AND METHODS
Subjects
study was approved by the University of North Carolina (Chapel Hill) School of Medicine Committee for the Protection of the Rights of Human Subjects, and all subjects provided writ¬ ten informed consent. We examined 92 subjects: 44 patients with major depression (28 men and 16 women) and 48 healthy con¬ trol subjects (29 men and 19 women; Table). Healthy control subjects and depressed patients were matched for mean age and sex as closely as possible. The age range for the depressed group was 18.6 to 46.6 years (mean±SEM, 30.8±1.2 years), and for the normal controls it was 18.9 to 45.6 years (28.2±1.0 years). All depressed subjects were studied through the University of North Carolina Mental Health Clinical Research Center (MHCRC). To include a broad range of depressed subjects, we studied subjects hospitalized in the MHCRC units at the University of North Carolina Neuropsychiatrie Hospital and the Dorothea Dix Hospital, Raleigh, NC, as well as four psychiatric outpatients examined on the University of North Carolina Gen¬ eral Clinical Research Unit. All depressed subjects met DSMIII-R criteria for major depression. Diagnoses were assigned by a board-certified research psychiatrist (D.L.E.), based on all available clinical data, including chart review of presenting signs and symptoms, psychiatric history, past treatment response, medical history, laboratory data, family history, and daily progress notes, as well as data generated from the Schedule for This
Affective Disorders and Schizophrenia-Lifetime version. Labo¬ ratory studies were performed in a double-blind fashion: diag¬ noses were assigned "blind" to all immune data, and laboratory data were obtained on coded blood samples. Depression ratings were obtained within 3 days of immune testing and consisted of
the 17-item Hamilton Rating Scale for Depression (HAM-D),28 which was administered by a psychiatric research nurse or fel¬ low of the MHCRC; the intraclass coefficient of correlation for these raters is R .91. The mean ± SEM depression ratings of the subjects with major depression and the normal control subjects are given in the Table. Healthy, normal control subjects were recruited by printed ad¬ vertisement and admitted to the University of North Carolina General Clinical Research Unit. The control subjects underwent the same systematic medical and psychiatric assessment as the depressed subjects, including physical examination and medical history, laboratory screening (complete blood cell count, serum electrolyte, urea nitrogen, creatinine, calcium, and protein val¬ ues, liver function tests, and thyroid function tests), the Schedule for Affective Disorders and Schizophrenia-Lifetime version, and the depression ratings. Control subjects were free of psychiatric illness, and all depressed and control subjects were free of any acute or chronic medical conditions known to affect immune function and were free, for at least 2 weeks before testing, of sub¬ stances or medications known to influence immune function. =
Procedures
Subjects took nothing by mouth starting at midnight before the drawing of blood the next morning. An intravenous line was
started at 8 am in an antecubital vein and maintained patent with slow drip of normal saline solution. To control for circadian ef¬ fects, peripheral blood for the immune measurements was ob¬ tained at 9:10 am. We obtained NK cell phenotype measures on all subjects to address hypothesis 1. To address hypothesis 2, NKA measurements were obtained on a subset of our sample (30 patients and 19 controls). The immune assays were performed in the Psychoneuroimmune Laboratory of the MHCRC. To reduce assay variance, fresh internal control samples were obtained from normal healthy controls and assayed for the monoclonal antibody determinations of NK cell populations; for the NK cell activity assays, internal control samples were cryopreserved following lymphocytapheresis.29 Ficoll-purified washed mononuclear cell populations were divided in aliquote and cryopreserved in the vapor phase of a liquid nitrogen freezer and thawed for each as¬ say. Each functional assay thus compared experimental subjects (either normal controls or depressed patients) with standardized frozen control samples. Monoclonal Antibody and Flow Cytometry Techniques.— Peripheral blood samples were collected into heparinized vac¬ uum (Vacutainer) tubes. The Leu series of monoclonal antibod¬ ies (Becton Dickinson, Mountain View, Calif) was used to measure the predominant reactivity as described previously.30 a
Briefly, mononuclear cells were partially purified by centrifugation through lymphocyte separation medium (Bionetics Labora¬ tory Products, Rockville, Md). Lymphocytes were collected by centrifugation and washed twice in RPMI1640 and adjusted to 1 107/mL. The cells were centrifuged in a centrifuge (SerofugeClay Adams Ine, Parsippany, NJ) and washed again in cold water-bovine serum albumin-azide-phosphate-buffered saline (pH 7.2). After the last wash step, the supernatant was poured off and 5 µ of the fluorescein-labeled monoclonal antibody re¬
was added for detection of cell surface markers. AntiLeu-7 and anti-Leu-HA monoclonal antibodies were used for their predominant reactivity with cells of the NK phenotype. A background control was used for nonspecific staining with fluorescein-labeled anti-IgGl. The cells were incubated with the monoclonal antibodies at 40°C for 45 minutes. Stained cells were washed twice with 1 mL of cold water-bovine serum albuminazide-phosphate-buffered saline, fixed by the addition of 1% paraformaldehyde, and stored in the dark at 4°C until examina¬ tion with a flow cytometer (FACScan, Becton Dickinson). Refer¬ ence ranges were determined by evaluation of normal peripheral blood controls. NKA Assay.—Natural killer cell cytotoxicity was measured in vitro using a chromium release assay described in detail else-
agent
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Fig 1.—Analysis ofcovariance adjusted forage revealed a significant difference between controls and patients with major depression (F[T, 731 7.46, P 008). Leu- values were logged for analysis and transferred back to original units (mean±SEM) for clarity of presen¬ tation. =
=
.
where.31 Briefly, following two washes in RPMI 1640 with 10% fetal bovine serum, varying dilutions of thawed effector cells from each subject (and internal control sample cells) were added (100 µ ) to triplicate U-bottom microtiter wells (Costar, Cam¬ bridge, Mass). Target cells were the NK-sensitive K562 tumor cell line labeled with 200 µ of sodium chromate-51 (10.8 MBq/mg; New England Nuclear, Boston, Mass) for 1 hour at 37°C. Following the labeling step, target cells were then washed three times in the above medium and counted, and a 100-µ aliquot (5000 cells) was added to wells containing effector cells. The mi¬ crotiter plates were then centrifuged for 5 minutes at 800 rpm and incubated for 18 hours in a humidified 95% air, 5% carbon diox¬ ide atmosphere. Following incubation, the plates were recentrifuged at 1200 rpm for 10 minutes. The supernatant from each well was collected and counted in a gammagraphic counter (LKB Co, Turku, Finland). Cytotoxicity was measured across five effectontarget cell (E:T) concentrations (200:1 to 12.5:1) for each subject. The percentage of lysis was determined using the
following equation: %Lysis (exp cpm-spont cpm)/(max cpm spont cpm) 100, where exp cpm was defined as the counts released into the medium by target cells when assayed =
-
with effector cells; max cpm was defined as known counts added in 5000 target cells; and spont cpm was defined as the counts re¬ leased by target cells in the presence of medium alone. Sponta¬ neous release was less than 5% of maximum release in all assays.
Statistical
Analysis
The Leu-7 and Leu-11 raw values were log transformed to sta¬ bilize group variance, and these logged values were then used as dependent measures in the statistical analyses. Analysis of covariance (ANCOVA) procedures, controlling for age effects,
Fig 2.—Analysis of covariance adjusted forage revealed a significant between controls (solid bars) and patients with major .015) but not in depression (open bars) in male (F[1,431 6.44, female subjects (FIT,271 =¡.57, P .22). Leu-H values were logged for analysis and transferred back to original units (mean±SEM) for clarity of presentation. difference
=
=
=
performed to examine the group differences between the patients with major depression and normal controls, as well as depression-control group differences for men and women sep¬ arately. The ANCOVAs for repeated measures were used to ex¬ amine group differences on NKA, where cytotoxicity was mea¬ sured at five E:T ratios. Correlational procedures were used to assess the relationship between severity of depression and the various immune measures, as well as the relationship between were
NK
phenotype and NKA.
All statistical tests
were
two-tailed.
RESULTS Pearson correlation analyses indicated that there was a signif¬ icant positive relationship between age and percentage of Leu-7 lymphocytes for all subjects in the study (r=.27, =.01). This result is consistent with other studies in the immunological lit¬ erature, showing that there is a positive correlation between age and percentage of Leu-7 lymphocytes from birth to senes¬ cence.11·32 In all subsequent analyses, age was used as a covariate to control for the effects of age on each immune measure and for consistency among data analyses. For clarity of presentation, the values presented in the figures for Leu-7 and Leu-11 are un¬
tagged values adjusted for age.
NK Cell Phenotypes in Peripheral Blood Patients with major depression were 27.4% lower in percent¬ age of Leu-11 lymphocytes compared with normal controls (F[l,73] 7.46, P=.008; Fig 1). As depicted in Fig 2, men with major depression were 31.6% lower in their percentage of Leu-11 lymphocytes when compared with male normal controls women with major depres¬ (F[l,43] 6.44, .015). sion also had a lower percentage of Leu-11 lymphocytes than fe=
=
=
Although
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Fig 3.—Analysis of covariance adjusted for age revealed a trend to¬ ward lower percentage of Leu-7 lymphocytes in patients with major depression compared with controls; however, statistical signifi¬ cance was not reached (F[¡, 861 3.62, .06). Leu-7 values were logged for analysis and transferred back to original units (mean±SEM) for clarity of presentation. =
male controls, this difference
was
=
not
(F[l,27] 1.57, P=.22).
Peripheral blood Leu-7 cell counts were lower (26.7%) in the group with major depression; however, this difference did not reach statistical significance (F[l,86] 3.62, .06; Fig 3). Figure 4 illustrates that men with major depression had 40% lower Leu-7 lymphocyte levels than male controls (F[l,53] 5.81, P= .02), but there was no difference between the women with major depression and female controls (F[l,30] 0.17, P=.68). =
=
=
Natural Killer Cell
difference between controls (solid bars) and patients with ma¬ .02) but not in jor depression (open bars) in male (F[¡,531 5.8¡, female subjects (F[¡, 30] 0.¡7, P .68). Leu-7 values were logged for =
=
analysis and transferred back clarity of presentation.
Activity
Natural killer cell activity data were available on a subset of the subjects in the lymphocyte phenotype analysis; there was no significant difference in the demographic and clinical character¬ istics of these subjects compared with the subjects in the pheno¬ type analysis. There was a graded lysis of K562 tumor target cells by effector cells of subjects from both diagnostic groups over the five E:T ratios tested, 200:1 to 12.5:1. A repeated-measures ANCOVA revealed a significant main effect of diagnosis (F[l,46] 6.66, =.01). Planned comparison of means analysis showed that subjects with major depression had lower NKA than control subjects at each E:T ratio, ranging from 25.1% lower at 200:1 (P= .02) to 46.7% lower at 12.5:1 (P= .03; Fig 5). As de¬ picted in Fig 6, when the major depression and control groups were divided by gender, men with major depression exhibited significantly lower NKA than male controls both when the data were collapsed across all of the E:T ratios (F[l,24] 7.82, P= .01) and at each of the five E:T ratios (31.7% to 51.9%; P= .006 to .02). Conversely, women with major depression had lower NKA but did not differ significantly from female controls at any of the five E:T ratios (5.0% to 33.6%; P= .38 to .82). =
=
=
=
to
original
values (mean±SEM) for
NK Cell Measures and HAM-D Scores
statistically significant
=
=
Fig 4.—Analysis of covariance adjusted for age indicated a signifi¬
cant
No significant relationship was noted between HAM-D sever¬ ity rating and either mean NKA or percentage of Leu-7 lympho¬ cytes in the depressed group. However, when the potential ef¬ fects of age and gender were controlled, the Pearson partial cor¬ relation between HAM-D rating and percentage of Leu-11 lymphocytes approached statistical significance (r=-.33, P= .06). Further analysis of the data by gender revealed a highly significant negative correlation between HAM-D rating and per¬ centage of Leu-11 lymphocytes in men with major depression (r= —.71, P= .0005), but not in women with major depression (r=.12, =.69). Natural killer cell activity was not related to HAM-D ratings in either men (r= .20, P= .49) or women (r= .15,
=.66).
Relationship of Leu-11
NK Cell
Phenotype to NKA
partial correlation (subtracting the variance ac¬ counted for by age and gender) confirmed the presence of the expected relationship between percentage of Leu-11 lympho¬ cytes and NKA in normal control subjects (Fig 7;r= .58, P= .03). Conversely, as seen in Fig 7, there was a negative relationship A Pearson
between percentage of Leu-11 cells and mean NKA among pa¬ tients with major depression (r= .36, P= .08). —
COMMENT These data demonstrate that NK effector cell popula¬ tions are reduced in depression, which confirms the find¬ ings from our earlier pilot study.30 Consistent with our prediction, significant reductions in Leu-11 lymphocyte counts were found in the group with major depression,
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200:1
100:1
25:1
50:1
12.5:1
EffectorTarget Cell Ratio of covariance adjusted for age showed significant differences (mean±SEM) in natural killer cell (NK) percent lysis for men at each effector.target cell ratio (F]¡,241 7.82, P .07): 200:¡ ( .006), ¡00:¡ ( .02), 50:¡ ( =.04), 25:1 ( .07), and ¡2.5:1 ( .07). This difference was not evidenced in women (FU, ¡91 0.30, P .59):200:¡ (P .83), ¡00:1 (P .73), 50:1 (P .38), 25:1 (P .65), and 12.5:1 (P .49). Solid triangles indicate male con¬ trols (n 70); solid circles, female controls (n 9); open triangles, men with major depression (n 77); and open circles, women with major depression (n 13).
Fig 6.—Analysis
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
lyse target cells. Killing capacity can be altered in sev¬ eral ways, including dysfunctional target recognition, impaired release of cytotoxic factors, decreased sensitiv¬ ity to regulatory cell signals, or altered regulatory cell function. Our results demonstrate that circulating NK cells labeled by Leu-11 monoclonal antibodies are reduced in depression, consistent with our prediction that de¬ creased Leu-11 cells would account for reductions in NKA. However, the expected positive correlation be¬ tween Leu-11 lymphocyte levels and NKA was highly significant in our normal control subjects18-25 but absent (and in fact negative) among depressed subjects. There¬ fore, reduced NKA in the depression group cannot be ex¬ plained solely on the basis of decreased circulating Leu-11 NK cells in the peripheral blood. These data suggest that alterations in both circulating levels of NK cells and their killing capacity are responsible for depression-associated reductions in NKA. When the data were analyzed by gender, there were striking depression-related gender differences for each of the three immune measures examined in this study. Male patients with major depression exhibited significant re¬ ductions in Leu-11 cell counts, NKA, and Leu-7 lympho¬ cyte counts compared with male controls. Conversely, women with major depression were not significantly dif¬ ferent from female controls for any of the three dependent variables studied. Thus, our hypothesis that Leu-11 lym¬ phocytes and NKA would be altered in depression was confirmed in men. We did not confirm this hypothesis in women. Our findings in women may be due to a weaker effect in women, a smaller female sample size, or possi¬ bly a floor effect in women. No study to date has explic¬ itly assessed whether major depression differentially af¬ fects immune function in male and female subjects. There is also a lack of preclinical and clinical data examining whether immunological responses to stress differ among male and female subjects. No previous study has assessed gender differences in NK cell phenotyping in major or
Fig 5.—Analysis of covariance adjusted for age showed that 30 sub¬ jects with major depression (open bars) had lower natural killer cell (NK) percent lysis (mean±SEM) than 79 controls (solid bars; Fit, 46] 6.6, P
