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Immunological response to stress in agoraphobia and panic attacks
768
BIOL
PSYCHIATRY
1986;21:768-774
Immunological Response to Stress in Agoraphobia and Panic Attacks Owen S. Surman, John Williams, David V. Sheehan, Terence B . Strom, Kenneth J. Jones, and James Coleman
The uuthors studied in vitro immune function us meusured by lymphocytic proliferative response to mitogen stimulation in 36 patients with agoraphobiu and punic ctttucks who were purticipating in a double-blind placebo-controlled psychopharmacological study of anxietv disorder. No signi~cant deference in immune status was observed between punit patients and healthy controls.
Introduction Data derived from several sources suggest that stress has significant impact on immune function. Early evidence for a link between stress and human susceptibility to infection is apparent in Dubos’ (1959) account of the parasitic destruction of the Irish potato crop in the mid-19th century and the subsequent spread of pulmonary tuberculosis. Working in the slums of Glasgow, Kissen (1958) reported an association between recent loss of a love object and the morbidity and mortality of pulmonary tuberculosis. Others (Heilig and Hoff 1982; Hull 1933; Ullman 1947) demonstrated that hypnotically induced stress reactivated herpes simplex infection in susceptible individuals; and warts, also of viral etiology, were observed to resolve in some patients treated with suggestion or hypnosis (Sinclair-Gieben and Chalmers 1958; Surman et al. 1973). A direct impact of psyche on immune defense was evident in the work of Black and coworkers ( 1963), who used hypnosis to reverse clinical evidence of delayed hypersensitivity in four individuals with allergy to tuberculin. Immediate-type hypersensitivity reactions were similarly reversed (Black 1963. 1969). The effect of stress on immune function was further documented in animal studies. Anterior hypothalmic lesions were shown to protect against lethal anaphylaxis in the rat (Luparello et al. 1964: Stein et al. 1967). Stress avoidance learning in laboratory animals was associated with altered susceptibility to herpes simplex virus (Rasmussen et al. 1957). passive anaphylaxis (Rasmussen et al. 1959). transplantation immunity (Wistar and Hil-
From the Department
of Psychiatry,
of Immunogenetics, Supported Address
by a grant reprint
Brigham from
requats
Harvard
the Upjohn tu Dr
Medtcal
and Womens Owen
School
Hospital.
and the Massachusetts
Boston,
General
Hospltai.
and Imm
the Department
MA.
Corporation. S. Surman.
Department
ol Psychlay.
Maaaachuettr
General
Hospital.
Boaton.
MA
02114. Adapted
from
related Received
0
1986 Society
preaentatwn
Anxiety September
of Biological
at the research
Disorders,
conlermcr
Boston,
November
16. 1985: revised
December
Psychiatry
on Bwlopul
Cons&ratlons
!n the Ettology
and Trcatmcnt
of Pamc-
6. 1983 23.
19X5
OM6-3223/86/$03
50
Immunological Response to Stress in Agoraphobia
BIOL PSYCHIATRY 1986;21:16~174
769
demann 1959), and tumor development in mice (Sklar et al. 1981) and poliomyelitis in monkeys (Marsh et al. 1963). Noise-induced stress in mice and rats has been associated with altered lymphocyte function (Folch and Waksman 1974; Monjan and Collector 1977). Ader and Cohen (1975, 1982) paired saccharin with cyclophosphamide in two taste aversion conditioning studies, which resulted, respectively, in depression of antibody formation in rats and modification of autoimmune disease in New Zealand hybrid mice. The first of these studies was replicated by Rogers et al. (1976, 1979). Evidence for afferent communication between the immune system and the ventromedial hypothalmus was presented by Besedovsky and Sorkin (1977). The mechanism for stress-induced immune suppression is most likely neuroendocrinologically mediated (Borysenko and Borysenko 1982). Lymphocytes are known to have receptors for a number of hormones. Agents that stimulate cyclic adenosine monophosphate (AMP), including beta-adrenergic catecholamines, histamine, and E series prostaglandins, inhibit the immunological actions of lymphocytes (Bourne et al. 1974). The effect of neuroendocrine pharmacological manipulation on immune function has been reviewed by a number of investigators (Yu and Clements 1976; Baker et al. 1977; Fugner 1977; Schwartz et al. 1977; Winchurch and Mardiney 1977; Pierpaoli and Maestroni 1978a,b; Gelfand 1979; Strom and Carpenter 1980). Recently, Crary et al. (1982) reported that epinephrine in physiological quantity altered proportions of T lymphocyte subsets. The authors suggested this as a possible factor in previously demonstrated immune suppressant effects of epinephrine. Other human studies have focused on stress-induced suppression of immune function (Locke 1982). Suppression of lymphocyte proliferative responses was demonstrated by Dorian et al. (1981) in psychiatric residents undergoing fellowship examination. Significantly higher mortality has been observed among widowed versus married middle-aged and older men (Helsing and Szklo 1981). Two studies have assessed immune function in this population. Bartrop et al. (1977) found a significant reduction in mitogen (phytohemagglutinin, PHA) induced lymphocyte proliferation in 26 prospectively studied bereaved men. Schleifer and coworkers (1983) prospectively studied 15 spouses of women with terminal breast cancer and found significant reduction of lymphocyte stimulation responses to mitogen [PHA, pokeweed mitogen (PWN), and concanavalin A (Con A)] in the first 2 months of bereavement. Kronfol et al. (1983) reported their study of lymphocyte proliferative response in 26 unmedicated depressed patients who showed markedly reduced responsiveness to PHA, PWM, and Con A when compared to 20 controls. Patients with agoraphobia and panic attacks present a natural model for stress. This anxiety disorder is in turn responsive to specific pharmacological intervention. To assess the possible relationship between agoraphobia with panic episodes and immune suppression, we prospectively studied a series of patients participating in a comparative study of psychopharmacological intervention with alprazolam, imipramine, phenelzine, and placebo.
Methods We postulated that patients meeting DSM-III criteria for “agoraphobia with panic attacks” would manifest lower in vitro mitogen-induced lymphotransformation responses than normal controls. We further postulated that agoraphobic patients whose panic attacks were treated with alprazolam, imipramine, or phenelzine would manifest an increased
770
BIOL PSYCHIATRY 1986;21:768-114
O.S. Surman et al.
response to posttreatment testing of mitogen-induced lymphocyte stimulation than patients treated with placebo. Forty-six patients were selected from a cohort of 125 patients in a double-blind placebocontrolled investigation of alprazolam, imipramine, and phenelzine in agoraphobics (Sheehan et al. 1985). Selection was based on scheduling arrangements established for the double-blind study. Thirty-six patients whose blood could be drawn between 3:30 and 6:30 PM, before and after 10 weeks of treatment with one of the three study drugs or placebo, were included in the final statistical analysis. Informed consent was obtained after the procedure had been fully explained. Twelve patients were men and 24 women. The mean age was 38.5 years, with a range of 22-59. Twenty patients underwent treatment between May and August 1982; 16 patients were in the double-blind protocol between October and December 1982. The mitogens used for in vitro immune study were PHA and PWM, which stimulate primarily T and B lymphocytes, respectively. All lectin solutions were prepared as stock solutions prior to initiation of the study. Peripheral blood was collected from patients or healthy volunteers into heparinized tubes that had been encoded for the double-blind study. The blood was diluted 1:3 in RPM1 1640 medium (MA Biproducts, Walkersville, MD) that had been buffered with 5 mM HEPES (isolation medium), and stored overnight at room temperature in 50-ml plastic tissue culture flasks (Falcon Co.). Mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia Co., Piscataway, NJ) density centrifugation and were washed three times in isolation medium containing 10% heat-inactivated fetal bovine serum (complete medium). One hundred microliters of the cell suspension was distributed in three sets of triplicate microtiter wells (Falcon Co.); thus, lo5 cells occupied each of nine wells. Each set of triplicate wells then received 100 p.1 of complete medium alone, 100 pl of 20 Fg/ml phytohemagglutinin-P (PHA-P) (Difco, Detroit, MI) in complete medium, or 100 ~1 of 20 kg/ml of pokeweed mitogen (PWM) (Sigma, St. Louis, MO) in complete medium. The cultures were incubated at 37°C in a 5% CO* incubator for 68 hr and were pulsed for 4 hr with 3H-thymidine (1 kc/well). The cells were harvested onto glass fiber strips (Mash II automated cell harvester), and 3H-thymidine incorporation was assessed by scintillation counting.
Results The lymphocyte (primarily T lymphocyte) response to PHA for the four treatment groups and healthy controls is presented in Table 1 in counts per minute (cpm X lo-‘). There
Table
1. Effect of Anxiety
Disorder
Treatment
on Lymphocyte
Response
to PHA
Treatment group Placebo Pretreatment cpm
X
SD n
X
Phenelzine
Control
89
117
I IO
I IO
139
102
127
I I?
II?
2s
19
IY
25
4’)
8
IO
9
7
118 IO-’
Posttreatment cpm
Imipraminc
Alprazolam
10-j 9
“Only two of the seven controls
were retested for PHA
resPo”w
BIOL PSYCHIATRY 1986;21:168-714
Immunological Response to Stress in Agoraphobia
771
Table 2. Effect of Anxiety Disorder Treatment on Lymphocyte Response to PWM Treatment group
Pretreatment cpm X 1W Posttreatment cpm x lo-3 SD n
Placebo
Alprazolam
Irnipramine
Phenelzine
Control
25
22
25
35
24
31
19
28
31
21”
12 9
14 8
10 10
13 9
13 7
“Only two of the seven controls were retested for PWM response.
is no significant difference in the response of anxious patients, pretreatment, and normal controls (F = 1.714, df = 4, p = 0.167). There is a significant increase in immune response following treatment (F = 4.821, df = 1, p = 0.035). However, the increased response is most pronounced among patients treated with placebo. Of the seven healthy volunteers, only two were retested after 10 weeks (mean 112 versus 110 cpm x 1W3). The response of lymphocytes (primarily B lymphocytes) to PWM for patients and controls is given in Table 2. No significant difference was found between patients and controls (F = 1.137, df = 4, p = 0.354), nor was there a significant change for the treatment factor (F = 0.216, df = 1, p = 0.5). A further three-way Analysis of Variance (ANOVA) was performed for response to PHA or PWM, Hamilton Depression score, and period of data collection (May-August 1982 versus October-December 1982). When we stratify on time of year, the increased response to PHA posttreatment remains approximately the same (p = 0.075 versus p = 0.035). The response to PWM is significant for the treatment factor (p = 0.012) among patients studied October-December. This difference is accounted for by a significantly lower pretreatment value (16.1 versus 32.6 cpm X 1C3) for the OctoberDecember patients versus those studied May-August. In separate analysis, a median split was used to form two groups of patients with high versus low Hamilton Depression score. There was no significant difference between these two groups for response to PHA or PWM in the ANOVA, nor was there a significant difference for the depression factor when the upper versus lower third scores on the Hamilton Depression Scale were analyzed. Stratifications of the Hamilton anxiety scores also failed to demonstrate a significant difference in immune response between those with scores on the lower versus the upper third of the scale. Anxiety scores decreased for all patients posttreatment (p = 0.001). Those treated with placebo had higher posttreatment Hamilton anxiety scores than those actively treated, but this difference did not reach statistical significance.
Discussion The data in this study do not support the hypothesis that phobic patients with panic attacks have impaired immune function as measured in vitro by lymphocytic proliferation in response to mitogen. The observed increase in response to PHA among anxious patients posttreatment was significant at a low level (p = 0.035). The increase in response was not selective for active pharmacological treatment versus placebo. Sheehan et al. (1985) found a significant improvement in anxiety scores among patients who received active
772
BIOL PSYCHIATRY 1986;21:768-774
O.S. Surman et al.
treatment (alprazolam, imipramine, or phenelzine) versus placebo. In the patient sample studied for in vitro immune function, there is a decrease in anxiety scores of actively treated versus placebo-treated patients, but the difference does not reach statistical significance, probably as a result of sample size. There was, however, a significant reduction in anxiety for all patients (p = 0.001) following treatment with drug or placebo. Factors influencing the outcome of mitogen stimulation include relative activity of the lectins and radioactive tracer material, presence of antibody in the fetal bovine serum, and nutritional status of the subject. These are unlikely to have influenced the basic findings of this study. However, there was a significant difference in response to PWM pretreatment, with lower scores evident among patients studied October-December. Reduction of anxiety scores posttreatment or adaptation of anxious patients to venipuncture could conceivably account for the observed increase in response to PHA posttreatment, but this interpretation is not consistent with the lack of significant difference in immune function between anxious patients pretreatment and healthy controls. High versus low Hamilton depression and anxiety scores were not associated with a significant impact on immune function. Our findings of normal in vitro immune function for anxious patients pretreatment relative to controls was unexpected in light of previous reports linking stress in humans with immune suppression. However, we must temper any negative findings in this study due to the low power attributable to small sample size. Fauman (1982) reviewed the effects of the central nervous system on immune function and concluded that adverse environmental events and psychiatric illness have an inconsistent effect on immune response. Possibly, the type of stress varies sufficiently in neurohumoral effect to account for a differential effect on immune function. Increased catecholamine production is thought to be an important factor in the effect of stress on immunity (Borysenko and Borysenko 1982; Crary et al. 1983). Stress-related production of corticosteroids has also been thought to be responsible for altered immune response. Stein et al. (1985) have found an adrenal-independent effect in rats studied for stress-related suppression of mitogen-induced lymphocyte stimulation. These authors and others (Borysenko and Borysenko 1982; Fauman 1982) point to a variety of neuroendocrine factors that play a mediating role in immunomodulation. Carr et al. (1985) studied immunological correlates in human volunteers undergoing lactate-induced anxiety. Untreated patients with panic disorder had increased baseline levels of catecholamines compared to normals. There was an absence of pituitary-adrenal activation in response to lactate. Growth hormone levels rose significantly in response to lactate in patients, but not in normals or patients receiving a placebo infusion. The finding of growth hormone as an apparent marker for anxiety may be relevant to the negative finding in the present report because of the potentiating effect of growth hormone on lymphocyte activation (Snow 1985).
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BIOL PSYCHIATRY 1986;21:768-774
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and immunity by cyclic
Carr DB, Sheehan DV, Surman OS, Coleman JH, Greenblatt DJ, Heninger GR, Jones KJ, Levine PH, Watkins DW (1985): Neuroendocrine correlates of lactate-induced anxiety and their response to chronic alprazolam therapy. Am J Psychiatry, in press. Crary B, Hauser S, Borysenko M, Kutz I, Hoban C, Weiner HL, Benson H (1982): Epinephrine induced changes in the distribution of lymphocyte subsets in peripheral blood of humans. J Immunol 131:1178-1181. Dorian BJ, Keysteon E, Garfmkel PE, Brown GH (1981): Immune mechanism logical stress. Psychosom Med 43:84, 1981.
in acute psycho-
Dubos R (1959): Mirage of Health: Utopias, Progress and Biological Change. New York: Harper and Row. Fauman MA (1982): The central nervous system and the immune system. Biol Psychiatry 17: 1459-1482. Folch H, Waksman BH (1974): The splenic suppressor cell: Activity of thymus dependent adherent cells: Changes with age and stress. J. Immunol 113:127-139. Fugner A (1977): Inhibition of antigen induced histamine release by beta adrenergic vivo. Int Arch Allergy Appl Immunol 54178-87.
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Helsing KJ, Szklo M (1981): Mortality and bereavement. Hull CJ (1933): Hypnosis and Suggestibility: Century, pp 272-274.
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Kissen DM (1958): Emotional Factors in Pulmonary Tuberculosis.
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Kronfol A, Silva J, Gredin J, Nembenski S, Gardner R, Carroll B (1983): Impaired lymphocyte function in depressive illness. Life Sci 33:241-247. Locke SE (1982): Stress, 4:49-58.
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Luparello TJ, Stein M, Park CD (1964): Effect of hypothalamic Physiol 207:911-914.
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Marsh JT, Lavender JF, Chang A, Rasmussen AF (1963): Poliomyelitis susceptibility after avoidance stress. Science 140: 1415-1416. Monjan AA, Collector 196:307-308.
MI (1977): Stress-induced
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immunosuppres-
Rogers MP, Dubey D, Reich P (1979): The influence of the psyche and the brain on immunity and disease susceptibility: A critical review. Psychosom Med 41: 147-163 Schleifer SJ, Keller SE, Lamerino M, Thornton JC, Stein M (1983): Suppression stimulation following bereavement. JAMA 250:374-377.
of lymphocyte
Schwartz A, Askenase PW, Gershon RK (1977): The effect of locally injected vasoactive amines on the elicitation of delayed type hypersensitivity. J Immunol 118: 159-165. Sheehan DVS, Claycomb B, Surman OS (1985): Comparative efficacy of imipramine, phenelzine, alprazolam, and placebo in the treatment of panic attacks associated with agoraphobia and the related phobic anxiety state. Am J Psychiatry, in press. Sinclair-Gieben AHC, Chalmers D (1959): Evaluation of treatment of warts by suggestion. ii:480-48 I . Sklar LS, Bruto V, Anisman H (198 1): Adaption to the tumor-enhancing Med 43:33 l-342.
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Snow CS (1985): Insulin and growth hormone function as minor growth factors that potentiate lymphocyte activation. J Immunol 135:7765-7785. Stein M, Schiavi RC, Luparello MS (1967): The hypothalamus Sci 164:464--169.
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Stein M, Keller SE, Schleifer SJ (1985): Stress and immunomodulation: and neuroendocrine function. J lmmunol 135:8273-8333.
The role of depression
Strom TB, Carpenter CB (1980): Cyclic nucleotides in immunosuppression macologic manipulation and in systems. Transplant Proc 12:304-3 10. Surman OS, Gottlieb SK, Hackett TP, Silverberg Arch Gen Psychiarp 28:439441.
E (1973): Hypnosis
neuroendocrine
phar-
in the treatment of warts.
Ullman M (1947): Herpes simplex and second degree bum induced under hypnosis. Am J. Psychiutn 103:828-830. Winchurch RA, Mardiney MR (1977): The effects of adrenergic agonists and blockers on antigeninduced DNA synthesis in vitro. Biomedicine 26:36-42. Wistar R, Hildemann WH (1959): Effect of stress on skin transplantation 131:159-160. Yu DT, Clements PJ (1976): Human lymphocyte lmmunol 25~472479.
subpopulations:
immunity in mice. Science
Effect of epinephrine.
C/in Exp
BIOL
PSYCHIATRY
1986;21:768-774
Immunological Response to Stress in Agoraphobia and Panic Attacks Owen S. Surman, John Williams, David V. Sheehan, Terence B . Strom, Kenneth J. Jones, and James Coleman
The uuthors studied in vitro immune function us meusured by lymphocytic proliferative response to mitogen stimulation in 36 patients with agoraphobiu and punic ctttucks who were purticipating in a double-blind placebo-controlled psychopharmacological study of anxietv disorder. No signi~cant deference in immune status was observed between punit patients and healthy controls.
Introduction Data derived from several sources suggest that stress has significant impact on immune function. Early evidence for a link between stress and human susceptibility to infection is apparent in Dubos’ (1959) account of the parasitic destruction of the Irish potato crop in the mid-19th century and the subsequent spread of pulmonary tuberculosis. Working in the slums of Glasgow, Kissen (1958) reported an association between recent loss of a love object and the morbidity and mortality of pulmonary tuberculosis. Others (Heilig and Hoff 1982; Hull 1933; Ullman 1947) demonstrated that hypnotically induced stress reactivated herpes simplex infection in susceptible individuals; and warts, also of viral etiology, were observed to resolve in some patients treated with suggestion or hypnosis (Sinclair-Gieben and Chalmers 1958; Surman et al. 1973). A direct impact of psyche on immune defense was evident in the work of Black and coworkers ( 1963), who used hypnosis to reverse clinical evidence of delayed hypersensitivity in four individuals with allergy to tuberculin. Immediate-type hypersensitivity reactions were similarly reversed (Black 1963. 1969). The effect of stress on immune function was further documented in animal studies. Anterior hypothalmic lesions were shown to protect against lethal anaphylaxis in the rat (Luparello et al. 1964: Stein et al. 1967). Stress avoidance learning in laboratory animals was associated with altered susceptibility to herpes simplex virus (Rasmussen et al. 1957). passive anaphylaxis (Rasmussen et al. 1959). transplantation immunity (Wistar and Hil-
From the Department
of Psychiatry,
of Immunogenetics, Supported Address
by a grant reprint
Brigham from
requats
Harvard
the Upjohn tu Dr
Medtcal
and Womens Owen
School
Hospital.
and the Massachusetts
Boston,
General
Hospltai.
and Imm
the Department
MA.
Corporation. S. Surman.
Department
ol Psychlay.
Maaaachuettr
General
Hospital.
Boaton.
MA
02114. Adapted
from
related Received
0
1986 Society
preaentatwn
Anxiety September
of Biological
at the research
Disorders,
conlermcr
Boston,
November
16. 1985: revised
December
Psychiatry
on Bwlopul
Cons&ratlons
!n the Ettology
and Trcatmcnt
of Pamc-
6. 1983 23.
19X5
OM6-3223/86/$03
50
Immunological Response to Stress in Agoraphobia
BIOL PSYCHIATRY 1986;21:16~174
769
demann 1959), and tumor development in mice (Sklar et al. 1981) and poliomyelitis in monkeys (Marsh et al. 1963). Noise-induced stress in mice and rats has been associated with altered lymphocyte function (Folch and Waksman 1974; Monjan and Collector 1977). Ader and Cohen (1975, 1982) paired saccharin with cyclophosphamide in two taste aversion conditioning studies, which resulted, respectively, in depression of antibody formation in rats and modification of autoimmune disease in New Zealand hybrid mice. The first of these studies was replicated by Rogers et al. (1976, 1979). Evidence for afferent communication between the immune system and the ventromedial hypothalmus was presented by Besedovsky and Sorkin (1977). The mechanism for stress-induced immune suppression is most likely neuroendocrinologically mediated (Borysenko and Borysenko 1982). Lymphocytes are known to have receptors for a number of hormones. Agents that stimulate cyclic adenosine monophosphate (AMP), including beta-adrenergic catecholamines, histamine, and E series prostaglandins, inhibit the immunological actions of lymphocytes (Bourne et al. 1974). The effect of neuroendocrine pharmacological manipulation on immune function has been reviewed by a number of investigators (Yu and Clements 1976; Baker et al. 1977; Fugner 1977; Schwartz et al. 1977; Winchurch and Mardiney 1977; Pierpaoli and Maestroni 1978a,b; Gelfand 1979; Strom and Carpenter 1980). Recently, Crary et al. (1982) reported that epinephrine in physiological quantity altered proportions of T lymphocyte subsets. The authors suggested this as a possible factor in previously demonstrated immune suppressant effects of epinephrine. Other human studies have focused on stress-induced suppression of immune function (Locke 1982). Suppression of lymphocyte proliferative responses was demonstrated by Dorian et al. (1981) in psychiatric residents undergoing fellowship examination. Significantly higher mortality has been observed among widowed versus married middle-aged and older men (Helsing and Szklo 1981). Two studies have assessed immune function in this population. Bartrop et al. (1977) found a significant reduction in mitogen (phytohemagglutinin, PHA) induced lymphocyte proliferation in 26 prospectively studied bereaved men. Schleifer and coworkers (1983) prospectively studied 15 spouses of women with terminal breast cancer and found significant reduction of lymphocyte stimulation responses to mitogen [PHA, pokeweed mitogen (PWN), and concanavalin A (Con A)] in the first 2 months of bereavement. Kronfol et al. (1983) reported their study of lymphocyte proliferative response in 26 unmedicated depressed patients who showed markedly reduced responsiveness to PHA, PWM, and Con A when compared to 20 controls. Patients with agoraphobia and panic attacks present a natural model for stress. This anxiety disorder is in turn responsive to specific pharmacological intervention. To assess the possible relationship between agoraphobia with panic episodes and immune suppression, we prospectively studied a series of patients participating in a comparative study of psychopharmacological intervention with alprazolam, imipramine, phenelzine, and placebo.
Methods We postulated that patients meeting DSM-III criteria for “agoraphobia with panic attacks” would manifest lower in vitro mitogen-induced lymphotransformation responses than normal controls. We further postulated that agoraphobic patients whose panic attacks were treated with alprazolam, imipramine, or phenelzine would manifest an increased
770
BIOL PSYCHIATRY 1986;21:768-114
O.S. Surman et al.
response to posttreatment testing of mitogen-induced lymphocyte stimulation than patients treated with placebo. Forty-six patients were selected from a cohort of 125 patients in a double-blind placebocontrolled investigation of alprazolam, imipramine, and phenelzine in agoraphobics (Sheehan et al. 1985). Selection was based on scheduling arrangements established for the double-blind study. Thirty-six patients whose blood could be drawn between 3:30 and 6:30 PM, before and after 10 weeks of treatment with one of the three study drugs or placebo, were included in the final statistical analysis. Informed consent was obtained after the procedure had been fully explained. Twelve patients were men and 24 women. The mean age was 38.5 years, with a range of 22-59. Twenty patients underwent treatment between May and August 1982; 16 patients were in the double-blind protocol between October and December 1982. The mitogens used for in vitro immune study were PHA and PWM, which stimulate primarily T and B lymphocytes, respectively. All lectin solutions were prepared as stock solutions prior to initiation of the study. Peripheral blood was collected from patients or healthy volunteers into heparinized tubes that had been encoded for the double-blind study. The blood was diluted 1:3 in RPM1 1640 medium (MA Biproducts, Walkersville, MD) that had been buffered with 5 mM HEPES (isolation medium), and stored overnight at room temperature in 50-ml plastic tissue culture flasks (Falcon Co.). Mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia Co., Piscataway, NJ) density centrifugation and were washed three times in isolation medium containing 10% heat-inactivated fetal bovine serum (complete medium). One hundred microliters of the cell suspension was distributed in three sets of triplicate microtiter wells (Falcon Co.); thus, lo5 cells occupied each of nine wells. Each set of triplicate wells then received 100 p.1 of complete medium alone, 100 pl of 20 Fg/ml phytohemagglutinin-P (PHA-P) (Difco, Detroit, MI) in complete medium, or 100 ~1 of 20 kg/ml of pokeweed mitogen (PWM) (Sigma, St. Louis, MO) in complete medium. The cultures were incubated at 37°C in a 5% CO* incubator for 68 hr and were pulsed for 4 hr with 3H-thymidine (1 kc/well). The cells were harvested onto glass fiber strips (Mash II automated cell harvester), and 3H-thymidine incorporation was assessed by scintillation counting.
Results The lymphocyte (primarily T lymphocyte) response to PHA for the four treatment groups and healthy controls is presented in Table 1 in counts per minute (cpm X lo-‘). There
Table
1. Effect of Anxiety
Disorder
Treatment
on Lymphocyte
Response
to PHA
Treatment group Placebo Pretreatment cpm
X
SD n
X
Phenelzine
Control
89
117
I IO
I IO
139
102
127
I I?
II?
2s
19
IY
25
4’)
8
IO
9
7
118 IO-’
Posttreatment cpm
Imipraminc
Alprazolam
10-j 9
“Only two of the seven controls
were retested for PHA
resPo”w
BIOL PSYCHIATRY 1986;21:168-714
Immunological Response to Stress in Agoraphobia
771
Table 2. Effect of Anxiety Disorder Treatment on Lymphocyte Response to PWM Treatment group
Pretreatment cpm X 1W Posttreatment cpm x lo-3 SD n
Placebo
Alprazolam
Irnipramine
Phenelzine
Control
25
22
25
35
24
31
19
28
31
21”
12 9
14 8
10 10
13 9
13 7
“Only two of the seven controls were retested for PWM response.
is no significant difference in the response of anxious patients, pretreatment, and normal controls (F = 1.714, df = 4, p = 0.167). There is a significant increase in immune response following treatment (F = 4.821, df = 1, p = 0.035). However, the increased response is most pronounced among patients treated with placebo. Of the seven healthy volunteers, only two were retested after 10 weeks (mean 112 versus 110 cpm x 1W3). The response of lymphocytes (primarily B lymphocytes) to PWM for patients and controls is given in Table 2. No significant difference was found between patients and controls (F = 1.137, df = 4, p = 0.354), nor was there a significant change for the treatment factor (F = 0.216, df = 1, p = 0.5). A further three-way Analysis of Variance (ANOVA) was performed for response to PHA or PWM, Hamilton Depression score, and period of data collection (May-August 1982 versus October-December 1982). When we stratify on time of year, the increased response to PHA posttreatment remains approximately the same (p = 0.075 versus p = 0.035). The response to PWM is significant for the treatment factor (p = 0.012) among patients studied October-December. This difference is accounted for by a significantly lower pretreatment value (16.1 versus 32.6 cpm X 1C3) for the OctoberDecember patients versus those studied May-August. In separate analysis, a median split was used to form two groups of patients with high versus low Hamilton Depression score. There was no significant difference between these two groups for response to PHA or PWM in the ANOVA, nor was there a significant difference for the depression factor when the upper versus lower third scores on the Hamilton Depression Scale were analyzed. Stratifications of the Hamilton anxiety scores also failed to demonstrate a significant difference in immune response between those with scores on the lower versus the upper third of the scale. Anxiety scores decreased for all patients posttreatment (p = 0.001). Those treated with placebo had higher posttreatment Hamilton anxiety scores than those actively treated, but this difference did not reach statistical significance.
Discussion The data in this study do not support the hypothesis that phobic patients with panic attacks have impaired immune function as measured in vitro by lymphocytic proliferation in response to mitogen. The observed increase in response to PHA among anxious patients posttreatment was significant at a low level (p = 0.035). The increase in response was not selective for active pharmacological treatment versus placebo. Sheehan et al. (1985) found a significant improvement in anxiety scores among patients who received active
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O.S. Surman et al.
treatment (alprazolam, imipramine, or phenelzine) versus placebo. In the patient sample studied for in vitro immune function, there is a decrease in anxiety scores of actively treated versus placebo-treated patients, but the difference does not reach statistical significance, probably as a result of sample size. There was, however, a significant reduction in anxiety for all patients (p = 0.001) following treatment with drug or placebo. Factors influencing the outcome of mitogen stimulation include relative activity of the lectins and radioactive tracer material, presence of antibody in the fetal bovine serum, and nutritional status of the subject. These are unlikely to have influenced the basic findings of this study. However, there was a significant difference in response to PWM pretreatment, with lower scores evident among patients studied October-December. Reduction of anxiety scores posttreatment or adaptation of anxious patients to venipuncture could conceivably account for the observed increase in response to PHA posttreatment, but this interpretation is not consistent with the lack of significant difference in immune function between anxious patients pretreatment and healthy controls. High versus low Hamilton depression and anxiety scores were not associated with a significant impact on immune function. Our findings of normal in vitro immune function for anxious patients pretreatment relative to controls was unexpected in light of previous reports linking stress in humans with immune suppression. However, we must temper any negative findings in this study due to the low power attributable to small sample size. Fauman (1982) reviewed the effects of the central nervous system on immune function and concluded that adverse environmental events and psychiatric illness have an inconsistent effect on immune response. Possibly, the type of stress varies sufficiently in neurohumoral effect to account for a differential effect on immune function. Increased catecholamine production is thought to be an important factor in the effect of stress on immunity (Borysenko and Borysenko 1982; Crary et al. 1983). Stress-related production of corticosteroids has also been thought to be responsible for altered immune response. Stein et al. (1985) have found an adrenal-independent effect in rats studied for stress-related suppression of mitogen-induced lymphocyte stimulation. These authors and others (Borysenko and Borysenko 1982; Fauman 1982) point to a variety of neuroendocrine factors that play a mediating role in immunomodulation. Carr et al. (1985) studied immunological correlates in human volunteers undergoing lactate-induced anxiety. Untreated patients with panic disorder had increased baseline levels of catecholamines compared to normals. There was an absence of pituitary-adrenal activation in response to lactate. Growth hormone levels rose significantly in response to lactate in patients, but not in normals or patients receiving a placebo infusion. The finding of growth hormone as an apparent marker for anxiety may be relevant to the negative finding in the present report because of the potentiating effect of growth hormone on lymphocyte activation (Snow 1985).
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