Yonago Acta medica 2004;47:1–5

Increased Activity of 2',5'-Oligoadenylate Synthetase in
Peripheral Blood Mononuclear Cells of Patients with Major Depressive Disorder
Takeshi Yamada, Kazufumi Ikuta*, Ryuzo Kawahara and Takeshi Sairenji*
Division of Neoropsychiatry, Department of Multidisciplinary Internal Medicine, School of
Medicine and *Division of Biosignaling, Department of Biomedical Sciences, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 683-8503 JapanThe aim of this study was to measure the activity of 2',5'-oligoadenylate synthetase (2',5'AS)
which is mainly induced by interferon (IFN)-α and -β in patients with major depression
and evaluate its relationship to the disease. 2',5'AS activity of 23 patients (male = 11,
female = 12) with major depression and of 29 normal control subjects (male = 15, female= 14) was measured in peripheral blood mononuclear cells (PBMCs) by radioassay. The
mean 2',5'AS activity in the PBMCs of the patients and that of the control subjects were1.10 ± 0.69% and 0.72 ± 0.51%, respectively. The activity in the patients was statisticallyhigher than that of the control subjects (P = 0.03). These results imply some abnormality
in the IFN-2',5'AS system of patients with major depression.
Key words: interferon; major depressive disorder; 2',5'-oligoadenylate synthetase

Major depressive disorder which is called major
depression commonly is an agnogenic psychiatricillness whose main symptoms are disturbed driveand mood. The patient of major depression often
shows loss of appetite, sleep disturbance, depressedmood, a feeling of sadness, helplessness, worthlessness and so on. Major depression and chronic fatigue syndrome (CFS) have some similar symptoms. However, it has been controversial whether
they have a common pathological mechanism or not(Krusei et al., 1989; Lane et al., 1991; Terman et al.,1998). It has been reported that 2',5'-oligoadenylate
synthetase (2',5'AS) activity within peripheral
blood mononuclear cells (PBMCs) was elevated inpatients with CFS (Suhadolnik et al., 1994; Vojdani
et al., 1998; Ikuta et al., 2003). 2',5'AS is known as
an interferon (IFN)-induced antiviral and anti-

proliferative gene product. 2',5'AS is mainly induced by IFN-α and -β, and 2',5'AS activity levels arethought to reflect the in vivo state of the IFN system(Sokawa et al., 1980; Shindo et al.,1989).
It is also known that IFN therapy for patientswith type B and C hepatitis and malignant tumors
induces 2',5'AS and sometimes causes serious fatigue and a depressive state. Because of these side
effects, patients are treated with antidepressants, or
IFN therapy is sometimes discontinued (Borden etal., 1998; Licinio et al., 1998; Valentine et al., 1998;
Musselman et al., 2001). The question is whether
or not and how much endogenous IFNs are involved in major depression. But it is difficult to evaluate IFN production, because cytokines including
IFN are released and consumed locally at the sitewhere the immune reaction occurs and they are

Abbreviations: AG Poly(I) Poly(C), agarose-polyriboinosinc acid-polyribocytidylic acid; 2',5'AS, 2',5'oligoadenylate synthetase; CFS, chronic fatigue syndrome; DEAE, diethylaminoethyl; DSM-IV, Diagnostic andstatistical manual of mental disorders, 4th ed.; 3H-2',5'-A, 3H-2',5'-oligoadenylate; HAMD, Hamilton depression
rating scale; IFN, interferon; PBMC, peripheral blood mononuclear cell; PMSF, phenylmethyl sulfonyl fluoride1

T. Yamada et al.

Table 1. Profiles and 2',5'AS activity of patients with major depression
and control subjects

on Diagnostic and statisticalmanual of mental disorders,
4th ed. (DSM-IV) criteria
Patient
Control subject
Statistics(American Psychiatric AsAge(year)
39.7 ± 10.9
37.5 ± 11.5
NS†sociation, 1994). Exclusion
Sex ratio(male/female)
11/12
15/14
NS‡
HAMD
20.8 ± 5.9
ND
criteria included acute or
Dose of antidepressants (mg)79.8 ± 86.00chronic infection, autoimDose of benzodiazepines (mg)15.8 ± 19.3
0munity, epilepsy, allergy,
Duration of illness(month)82.0 ± 79.0—
neoplasia, endocrine disDuration of treatment (month)38.4 ± 54.002',5'AS activity(%)
1.10 ± 0.69
0.72 ± 0.51
P < 0.05†
ease, CFS or other acute
Data are mean ± SD, except the sex ratio.
physical diseases for both
ND, not determined; NS, no significant difference.
groups. Subjects were ex† Student’s t-test was used for statistical analysis.
2
cluded if they fulfilled any‡ χ test was used for statistical analysis.
additional axis I or axis II
DSM-IV diagnosis for the
seldom detectable in peripheral blood (De Groote etpatients and any axis I or axis II DSM-IV diagnosisal., 1992). 2',5'AS activity has been measured alterfor control subjects, respectively. These illnesses
natively to IFN-α and -β activity (Sokawa et al.,were ruled out by clinical interview, physical exam1980; Schattner et al., 1981; Uno et al., 1998). IFNination and comprehensive laboratory work. For
α and -β in sera of patients with major depression
the assessment of depressive severity, psychopathhave never been evaluated in a proper manner. The
ology was quantified by the Hamilton depression
aim of this study is to measure the activity ofrating scale (HAMD) 21-item version at the time of
2',5'AS in PBMCs of patients with major depresblood sampling (Hamilton, 1960). The meansion and evaluate its relationship to the disease.
HAMD score of the patients was 20.8 ± 5.9 ranging
This is the 1st report regarding 2',5'AS activity infrom 15 to 36. Most of them were on antideprespatients with major depression, as far as we know.
sants such as clomipramine, maprotiline and fluvoxamine, while some were on benzodiazepines suchas diazepam and flunitrazepam. The mean daily
Materials and Methods
doses of antidepressants and benzodiazepins ineach patient were 79.8 ± 86.0 mg (imipramine
equivalents; range: 0–265 mg) and 15.8 ± 19.3 mg
Subjects(diazepam equivalents; range: 0–81.5 mg), respecThis study was permitted by the Medical Ethics
tively. Inagaki’s conversion tables for antidepresCommittee at the Tottori University Faculty ofsants and benzodiazepines were used (Inagaki et al.,
Medicine. Informed consent was obtained from1999). The mean durations of illness and treatment
every patient and healthy control subject with awere 82.0 ± 79.0 months ranging from 1 to 324
detailed explanation of the purpose and goal of themonths and 38.4 ± 54.0 months ranging from 0.3 tostudy.
192 months, respectively (Table 1).
Eleven male and 12 female patients (meanage: 39.4 ± 10.9 years ranging from 20 to 53 years)
2', 5'AS activity assay
with major depression and 15 male and 14 female
healthy subjects (mean age: 37.5 ± 11.5 years rangPeripheral blood was collected between 0900 anding from 20 to 59 years) as controls participated in
1200 from each subject and kept on ice. PBMCs
this study. There was no significant difference bewere isolated within 30 min by Ficoll-Conray
tween the patient and the healthy control groups
centrifugation (Boyum, 1968). The 2',5'AS activity
concerning age and sex ratio (Table 1). The clinicalwas measured as previously described (Sokawa etdiagnosis for major depressive disorder was basedal., 1980; Ikuta et al., 2003). For the assay, buffer A22',5'AS activity in major depression

beads were incubated at 33˚C for 2 h with 20 µL of
buffer B containing 0.5 mM cold ATP and 0.4 µCi
of 3H-ATP (Moravek Biochemicals, LaBrea, CA).
The sample was suspended in 1 mL of buffer C andthe supernatant was collected by centrifugation at
5,000 rpm for 1 min. The product [3H-2',5'-oligoadenylate (3H-2',5'-A)] was absorbed on 50 µL ofdiethylaminoethyl (DEAE) Sepharose CL-6
(Amersham Pharmacia Biotech) pre-equilibrated
with buffer C. The DEAE Sepharose was washed 5times with 1 mL of buffer C and then suspended in
500 µL of buffer D to solubilize 3H-2',5'-A. The
radioactivity of 3H-2',5'-A in each sample was measured in a scintillator (ACS2: Amersham Pharmacia
Biotech) by a scintillation counter. The 2',5'AS activity was expressed as a percentage of the incorporated 3H-ATP by the enzyme in each sample. It
took about 6 h from equilibration of AG Poly(I)Poly(C) to the measure of radioactivity. The intraand inter-assay coefficients for 2',5'AS activity
were 4.8% and 16.6%, respectively.

[10 mM HEPES (adjusted to pH 7.5 by 1 M KOH),
3 mM magnesium acetate, 0.3 mM EDTA and 10%
glycerol], buffer B (50 mM KCl in buffer A), buffer
C [0.2 mM phenylmethyl sulfonyl fluoride (PMSF)
and 7 mM 2-mercaptoethanol in buffer B] andbuffer D (400 mM KCl in buffer A) were preparedand stocked at 4˚C until use.
The procedure of 2',5'AS activity assay wasdone at room temperature, if not mentioned otherwise. The 106 PBMCs were washed with phosphatebuffered saline and were suspended in 40 µL of
Triton extraction buffer (0.5% Triton X-100, 7 mM
2-mercaptoethanol and 1 mM PMSF in buffer B).
The sample was mixed well by pipetting, and thecell extract of the sample was obtained by centrifugation at 14,000 rpm for 5 min at 4˚C. The cell
extract was stored at –80˚C and used for the 2',5'AS
activity assay within 1 week: 20 µL of the cell extract was added to 25 µL of agarose-polyriboinosincacid-polyribocytidylic acid [AG Poly(I) Poly(C)type 6: Amersham Pharmacia Biotech, Tokyo,
Japan] and put on ice for 1 h to activate 2',5'AS in
the cell extract. The AG Poly(I) Poly(C) was equilibrated with buffer C before use. Then, 1 mL ofbuffer C was added to the mixture which contains
2',5'AS with AG Poly(I) Poly(C) and followed bycentrifugation at 5,000 rpm for 1 min. The washed

Statistical analysis
Statistical differences in age and 2',5'AS activity
between the 2 groups were estimated by Student’s ttest and that of sex distribution was estimated by theχ2 test. Statistical difference in 2',5'AS activity
between all male and female subjects was estimatedby Student’s t-test. Coefficients of 2',5'AS activities with ages, HAMD scores, dose of antidepressants, dose of benzodiazepines, duration of illness
and duration of treatment were estimated by
Pearson’s coefficient. Differences were considered
to be significant when P values were less than 0.05.

(%)
3.0

ATP incorporation

2.5

2.0

1.5

1.0

Results
0.5

2',5'AS activity was assayed in PBMCs of patientswith major depression and healthy control subjects(Fig. 1). The mean 2',5'AS activity of ATP incorporation rate in patients with major depression andthat of the healthy control subjects were 1.10 ±
0.69% and 0.72 ± 0.51%, respectively. 2',5'AS activity in the patients was statistically higher thanthat of the healthy control subjects (P = 0.03).

0Patient

Control

Fig. 1. The activity of 2',5'AS in PBMCs of patients withmajor depression and normal control subjects. Each dot
shows the 2',5'AS activity for each subject. The mean
2',5'AS activity (± SD) in patients and that of the control
subjects were 1.10 ± 0.69% and 0.72 ± 0.51%, respectively.

3

T. Yamada et al.

No significant correlation between 2',5'AS
activities in PBMCs and age, HAMD scores, doseof antidepressants, dose of benzodiazepines, duration of illness or duration of treatment was observedin the patients, as well as no significant correlation
between 2',5'AS activities in the PBMCs and ages
in the healthy control subjects. There was also no
significant sex difference of 2',5'AS activity in the
PBMCs of all subjects.

pression (Bode et al., 1993). Our results may suggest the involvement of viral infections in patientswith major depression.
Elevation of 2',5'AS activity in PBMCs of patients with major depression may be explained byanother possibility: major depression is usually accompanied by decreased natural killer cell functions (Stein et al., 1991), and 2',5'AS might be activated as the alternative defense mechanism. The
elevation of 2',5'AS activity in CFS could be a primary abnormality, because 37 kDa RNase L which
is a truncated form of the native 83 kDa RNaseL isdetected in patients with CFS but not in patientswith major depression nor in healthy controls(Suhadolnik et al., 1997; De Meirleir et al., 2000;
Demettre et al., 2002). RNase L is an enzyme whichis activated with the 2',5'AS system to degrade
cellular and viral single strand RNA. Thus there
seems to be a different mechanism between major
depression and CFS according to the elevation of
2',5'AS activity.
Most of our patients with major depression
had antidepressants and benzodiazepines. Recently, it has been reported that antidepressantshave a critical effect on the cytokine production of
IFN-γ (Maes et al., 1999; Kubera et al., 2001). Even
though we observed no significant correlation between 2',5'AS activity and those drugs in the patients, the possibility that antidepressants or benzodiazepines up-regulate 2',5'AS activity remains. In
order to study the effects of those drugs on 2',5'AS
activity, 2',5'AS activity in patients with major depression must be assayed before and after treatment
with the drugs.
In conclusion, our study demonstrates the significant elevation of 2',5'AS activity in major depression, which implies the involvement of the
IFN-2',5'AS system. The possible mechanisms of
up-regulation of the enzyme remain to be studied.

Discussion
We found that 2',5'AS activity was significantly
elevated in PBMCs of the patients with major
depression to that of the healthy control subjects.
A depressive state is found during IFN therapy
as a side effect. IFN therapy which induces 2',5'AScauses not only a central nervous system-depressive
state but also somatic symptoms such as joint pain,
muscle pain, fatigue and so on (Borden et al., 1998).
Major depression is sometimes accompanied bythose somatic symptoms, which are thought to be
one of the aspects of major depression. The question was whether or not and how much endogenous
IFNs are involved in major depression. IFN-α and-β in sera of patients with major depression havenever been evaluated in a proper manner. Our
results imply the involvement of IFN-α and -β inmajor depression.
Elevation of 2',5'AS activity in PBMCs ofpatients with CFS has been reported (Suhadolnik etal., 1994; Vojdani et al., 1998; Ikuta et al., 2003). In
our study, 2',5'AS activity in PBMCs were also
elevated in the patients with major depression.
Even though CFS and major depression are mutually exclusive by strict diagnostic definition, there aremany reports which indicate clinical overlapping orsome connections between CFS and major depression (Krusei et al., 1989; Lane et al., 1991; Fukuda
et al., 1994; Terman et al., 1998).
2',5'AS activity especially has been noticeablewith viral infections (Schattner et al., 1981; BuffetJanversse et al., 1983; Shindo et al., 1988). For the
relation between virus and major depression, Borna
disease virus antibody has been reported to be
detectable within sera in patients with major de-

Acknowledgments: This study was supported by a Grant
in Aid for Scientific Research from Special Coordination Funds for Promoting Science and Technology from
the Ministry of Education, Culture, Sports, Science andTechnology, the Japanese Government.

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2',5'AS activity in major depression

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Received December 5, 2003; accepted January 8, 2004
Corresponding author: Takeshi Yamada

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