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Panic disorder and immunity: Few effects on circulating lymphocytes, mitogen response, and NK cell activity
BRAIN, BEHAVIOR, and IMMUNITY Brain, Behavior, and Immunity 16 (2002) 698–705 www.academicpress.com
Panic disorder and immunity: Few effects on circulating lymphocytes, mitogen response, and NK cell activity Steven J. Schleifer,* Steven E. Keller, and Jacqueline A. Bartlett Department of Psychiatry, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA Received 24 July 2001
Abstract Altered immune measures are commonly found in major depression (MD), however, less is known about the immune system in anxiety disorders. We examined quantitative and functional in vitro immune measures in patients with panic disorder (PD), which is often comorbid with MD. Fourteen otherwise healthy medication-free adults (ages 23–49; 11 female) meeting SCID-UP DSM-IIIR criteria for PD with agoraphobia and without current MD, were compared with 14 subjects free of PD, MD, or other major psychiatric disorders, matched by gender, age, and racial background. PD was associated with decreased percentage (p < :03) and total (p < :03) circulating CD19+ B lymphocytes, but no differences in other enumerative lymphocyte measures. Mitogen responses (Con A, PHA, PWM) did not differ except for possibly decreased PHA in PD (p < :06). NK cell activity did not differ between PD and control subjects. The few immune measure changes in PD contrast with those found in MD, providing further evidence for the specificity of immune changes in psychiatric disorders. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Panic disorder; Immunity; Lymphocytes; Mitogens; Phytohemagglutinin; NK cells; B cells; Anxiety disorders; Agoraphobia
1. Introduction An association between depressive disorders and altered immunity has been described in many studies (Herbert & Cohen, 1993a). Less is known about the immune system in panic disorder (PD), which shares some biological, clinical, and therapeutic characteristics with affective disorders, is often comorbid with major depression (MD), but appears to be a separate entity (Abelson & Curtis,
*
Corresponding author. Fax: 1-973-972-8305. E-mail address: [email protected] (S.J. Schleifer).
1996; Ballenger, 1999; Gorman, Kent, Sullivan, & Coplan, 2000; Kessler et al., 1998; Weissman et al., 1993). The possible isomorphism of panic disorder and increased stress reactivity (Gorman et al., 2000) further suggests the possibility of immune system changes in PD such as those regularly found following stress exposure (Herbert & Cohen, 1993b). Studies from our group found that comorbid anxiety disorders in depressed patients were associated with immune measures that differed from those found with MD alone (Andreoli et al., 1992). Studies of immunity in PD without MD have been limited and have tended to focus on a single
0889-1591/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 8 8 9 - 1 5 9 1 ( 0 2 ) 0 0 0 2 2 - 3
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immune dimension. To investigate whether persons with PD show immune changes comparable to those found in depressive disorders, we compared medication-free patients without medical disorders who met criteria for PD and not MD with matched subjects without a history of panic or depressive disorder. PD and non-PD subjects were compared with respect to the number and distribution of circulating leukocytes, lymphocytes and lymphocyte subsets, mitogen induced lymphocyte proliferation, and natural killer (NK) cell activity.
2. Method The study was approved by the Institutional Review Board of UMDNJ-New Jersey Medical School. Informed consent was obtained from all subjects. Eighteen subjects and 18 matched controls over 18 years of age were recruited, of whom 14 pairs were retained in the current analyses (four PD patients were found on further evaluation to meet criteria for current MD or medical exclusionary criteria). The PD subjects were recruited primarily from the outpatient Panic Disorder and Phobia Clinic at Hackensack University Medical Center, and from other clinical programs of the UMDNJ-New Jersey Medical School, Newark, NJ. A medical history, review of systems and vital signs were obtained along with standard laboratory screening. Potential subjects were excluded if they had evidence for diseases likely to have substantial effects on immunity (e.g., neoplastic, endocrine or immune disorders, acute infectious processes) or if they were taking medications with known immunologic effects. No subjects were taking psychotropic medications at study entry (and for at least several weeks beforehand) with the exception of three who were taking diazepam or lorazepam on an as needed basis. PD subjects were each studied on the same day as their matched control, who met the same medical inclusion/ exclusion criteria and had no history of MD, PD, or other major psychiatric disorders. Control subjects were matched for age, sex, and race and were recruited from UMDNJ and Hackensack University Medical Center personnel and from surrounding communities. Diagnostic assessments were made utilizing the SCID-UP, a semi-structured diagnostic instrument designed to generate DSMIII-R psychiatric diagnoses, and modified to include more extensive data in relation to panic disorder (Spitzer,
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Williams, & Gibbon, 1986; American Psychiatric Association, 1987). The Hamilton anxiety scale (HARS) (Hamilton, 1959) and the Hamilton depression rating scale (HDRS) (Hamilton, 1967) were utilized. All assessments were made by trained interviewers, who had achieved interrater reliability (>0.80) for DSM-IIIR diagnoses (Kappa) and for the HAS and HDRS (intraclass correlation coefficient). Upon completion of each interview, 30 ml of blood were collected for immune studies. Control subjects were compensated for their time, effort, and expenses. Subjects were studied, in general, during the late morning or early afternoon. All data were collected with interviewers and laboratory technicians ‘‘blinded’’ to the maximum extent possible: Technicians received blood samples with only a sequential number for identification and interviewers were blind to the biological data. Immune assays were carried out the same day as venipuncture. Blood samples were collected into a heparinized syringe (preservative-free heparin). Total white blood cell and differential counts were performed by standard techniques. Lymphocytes were separated from whole blood by centrifugation on a Ficoll–Hypaque gradient (Pharmacia Fine Chemicals, Piscataway, NJ). Aliquots of these lymphocytes were utilized in the in vitro assays. Lymphocyte subsets [T (CD3+), B (CD19+), NK (CD56+), T4 (CD4+), T8 (CD8+), T4 inducer of help (CD29+), T4 inducer of suppression/naive cells (CD45RA+), and activated T cells (HLADR+)] were assessed by flow cytometry using a battery of paired (FITC and PE) monoclonal antisera (Coulter Cytometry, Hialeah, FL). The proliferative response of isolated peripheral blood lymphocytes to mitogens was assessed according to standard techniques (Keller et al., 1981). Two peak doses were utilized for phytohemagglutinin (PHA; Wellcome Reagents, Beckenham, UK), Concanavalin A (Con A; Calbiochem, San Diego, CA), and pokeweed mitogen (PWM; GIBCO, Gaithersburg, MD). Lymphocyte stimulation data are expressed as counts per minute (cpm) in the stimulated cultures minus the cpm in the unstimulated cultures (D cpm). D cpm data were log transformed for all analyses to stabilize the variance. NK cell activity (NKCA) was assessed utilizing K562 target cells according to standard methods as modified by the investigators (Georgescu & Keller, 1987). Analysis of variance (ANOVA) for repeated measures was used to test overall mean differences between the PD and control groups for the
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mitogen assays (mitogen dose as the repeated measure) and NK cell function (effector:target cell ratio as the repeated measure) and paired t tests were used to test for differences in the other immune variables. For exploratory analyses assessing the contribution of potential modulating variables, covariance analyses, and hierarchical multiple regression analyses were used.
3. Results The characteristics of the PD and matched non-PD subjects are shown in Table 1. The age range for the PD subjects was 23–49. The female/ male ratio of 3:1 is in keeping with the higher prevalence of PD in women. HARS scores ranged from 9 to 23. HDRS scores (1–23) ranged from levels associated with none to moderate affective
Table 1 Subject characteristics: Means SD (range) Variable
PD subjects Matched (n ¼ 14) controls (n ¼ 14)
Age
37:5 8:2 (23–49)
37:2 7:8 (24–51)
Gender Male Female
3 11
3 11
Race African–American Caucasian
1 13
1 13
Hamilton depression scale 7:6 5:7 (1–23) (HDRS)
0:9 1:2 (0–4)
Past MDD Yes No
9 5
0 14
Tobacco users Packs/day past 2 years Packs/day past 2 weeks
8 0:54 0:75 0:36 0:63
7 0:04 0:13 0:07 0:27
Hamilton anxiety scale (HARS)
14:0 4:1 (9–23)
1:3 2:4 (0–8)
Alcohol: Drinks/day Past 2 years Past 24 h
3:1 3:5 0:5 1:4
1:8 2:3 0:7 1:5
1:9 1:6
2:7 1:9
Caffeinated beverages past 24 h p < :01. ** p < :05. *
symptoms. Nine of the 14 PD subjects met criteria for prior major depression. PD patients reported significantly more tobacco use, but did not differ significantly in the use of alcohol or caffeine. All PD subjects met criteria for agoraphobia. 3.1. Enumerative measures As seen in Table 2, comparisons of the percentages of leukocytes, lymphocytes, and lymphocyte subsets for the PD patients and their matched controls showed decreased circulating percentages of (CD19+) B cells (p < :025). There were no other differences between the PD patients and their matched controls in the distribution of circulating leukocytes and lymphocyte subsets. Comparison of the number of circulating leukocytes and lymphocytes for the PD patients and their matched controls (Table 3) revealed, similarly, decreased circulating B cells (p < :03) and no differences in the other circulating leukocytes, lymphocytes, or lymphocyte subsets. 3.2. Functional measures As seen in Fig. 1, lymphocyte stimulation responses for the mitogens Con A, PHA, and PWM appeared to be comparable for patients with PD and their matched controls. Repeated measures ANOVA revealed no significant differences for any of the mitogens, except for a possibly decreased overall response to PHA in the PD subjects: Con A (group: F ¼ 0:36; df ¼ 1; 11, p n.s.; dose: F ¼ 15:06; df ¼ 1; 11; p < :005; group X dose: F ¼ 0:07; df ¼ 1; 11, p n.s.); PHA (group: F ¼ 4:91; df ¼ 1; 7; p < :06; dose: F ¼ 34:61; df ¼ 1; 7; p < :001; group X dose: F ¼ 0:16; df ¼ 1; 7, p n.s.); PWM: (group: F ¼ 1:67; df ¼ 1; 9; p n.s.; dose: F ¼ 0:53; df ¼ 1; 9, p n.s.; group X dose: F ¼ 0:39; df ¼ 1; 9, p n.s.). As seen in Fig. 2, NKCA was comparable for PD subjects and their matched controls. ANOVA for repeated measures on NKCA revealed no significant PD or PD by effector:target ratio effects (group: F ¼ 0:01; df ¼ 1; 11, p n.s.; E:T ratio: F ¼ 81:44; df ¼ 2; 10; p < :0001; group X E:T ratio: F ¼ 0:94; df ¼ 2; 10, p n.s.).
4. Discussion The present study of otherwise healthy, medication free patients meeting criteria for PD with agoraphobia, but not current MD, showed no
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Table 2 Peripheral leukocyte measures: Panic disorder subjects and matched controls (cells as percent of leukocytes/lymphocytes SD) Variable
Panic disorder subjects
Matched control subjects
t
Paired n
p
Lymphocytes Granulocytes Monocytes T lymphocytes B lymphocytes CD4+ lymphocytes CD8+ lymphocytes CD29+ lymphocytes (inducers of help) CD45RA+ lymphocytes (inducers of suppression) CD56+ lymphocytes (NK cells) HLA-DR+ lymphocytes (activated T cells)
31:2 8:3 61:1 8:2 7:17 2:42 83:4 4:6 10:6 3:5 50:9 8:3 21:9 4:0 23:2 5:9 22:9 8:9 5:08 4:50 4:70 2:12
36:2 5:8 57:3 5:9 6:03 0:82 81:7 4:2 13:9 3:9 50:2 6:9 22:5 5:1 23:3 4:5 21:2 5:6 3:88 2:13 5:46 4:15
1:58 1.28 1.62 1.38 2:58 0.32 0:38 0:08 0.77 0.80 0:76
11 11 11 14 14 14 14 8 9 14 14
n.s. n.s. n.s. n.s. <.025 n.s. n.s. n.s. n.s. n.s. n.s.
Table 3 Peripheral leukocyte measures: Panic disorder subjects and matched controls (mean cells/ml 106 SD) Variable
Panic disorder subjects
Matched control subjects
t
Paired n
p
Leukocytes Lymphocytes Granulocytes Monocytes T lymphocytes B lymphocytes CD4+ lymphocytes CD8+ lymphocytes CD29+ lymphocytes (inducers of help) CD45RA+ lymphocytes (inducers of suppression) CD56+ lymphocytes (NK cells) HLA-DR+ lymphocytes (activated T cells)
7:20 2:60 2:33 0:72 4:81 1:94 0:53 0:16 1:96 0:63 0:25 0:11 1:20 0:47 0:50 0:19 0:52 0:14 0:59 0:37 0:10 0:11 0:11 0:05
7:19 1:72 2:70 0:97 4:16 1:08 0:44 0:16 2:24 0:82 0:36 0:13 1:35 0:46 0:60 0:33 0:66 0:20 0:62 0:31 0:11 0:07 0:14 0:08
0.01 1:01 1.12 1.31 0:91 2:55 0:80 0:94 1:59 0:27 0:22 0:98
14 11 11 11 11 11 11 11 8 9 11 11
n.s. n.s. n.s. n.s. n.s. <:03 n.s. n.s. n.s. n.s. n.s. n.s.
evidence for significant differences from matched controls in the number or distribution of leukocytes, lymphocytes, or lymphocyte subsets, lymphocyte proliferative responses to T or B cell mitogens, or NK cell activity. The exceptions were a decrease in the proportion and number of CD19+ cells (B lymphocytes) and a possible decrease in PHA response. Other studies have investigated the proportion of CD19+ cells in PD, including two that found no differences from controls (Marazziti et al., 1992; Manfro et al., 2000) and two that found increased CD19+ cells in PD patients (Perini et al., 1995; Rapaport, 1998). These reports included study samples that appear to have been demographically and clinically similar to the present sample, with the exception of the small proportion of males in the current study and our only including PD
subjects with agoraphobia. Future studies are needed to clarify’ factors such as gender and disorder subtype and tobacco and other substance use (see below) that may interact in the apparent association between PD and circulating B cells. Studies of the distribution of lymphocytes and other lymphocyte subsets in PD have, in general, not replicated several single reports of differences from non-PD subjects. Single studies have reported decreased circulating total lymphocytes (Ramesh, Yeragani, Balon, & Pohl, 1991), decreased CD3+ (total T) cells (Perini et al., 1995), and decreased CD4+ (T helper) cells (Marazziti et al., 1992). For each of these observations, at least one other study (e.g., Manfro et al., 2000; Rapaport, 1998) found no significant differences between PD patients and controls, and the current study similarly found no differences in these
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Fig. 1. Response to PHA, Con A, and Pokeweed Mitogen (log transformed; means SEM) for panic disorder subjects (PD) and matched controls (Ctrl).
Fig. 2. NK cell activity (% cytotoxicity) (means SEM) for panic disorder subjects (PD) and matched controls (Ctrl).
measures. One study found increased total leukocytes in PD (Ramesh et al., 1991) and one increased NK cells (Rapaport, 1998), findings that were also not confirmed in other studies (e.g., Manfro et al., 2000; Marazziti et al., 1992) including the present report. There are now two
reports of increased HLA-DR+ cells in PD (Perini et al., 1995; Rapaport, 1998), an effect that, as noted (Rapaport, 1998), may have been a consequence of the increased B cells found in the PD subjects rather than an indication of increased activated T cells. Correspondingly, both the current report and that of Marazitti et al. (1992), which did not find increased B cells, found no increase in HLA-DR+ cells. Mitogen responses, a measure of the functional capacity of T or B cells to proliferate in response to a nonspecific challenge, has been found to be altered in subgroups of patients with depressive disorders and following life stress (Herbert & Cohen, 1993a,b). The current findings, showing possibly decreased PHA in PD (p < :06), indicate a need for further investigation with larger samples considering two prior reports of no PHA differences (Brambilla et al., 1992; Surman et al., 1986) but one of decreased PHA response in PD (Koh & Lee, 1998). The failure to detect differences in PWM response are consistent with the one previous report in PD (Surman et al., 1986). The lack of differences for Con A, which apparently has not been assessed previously, provides additional evidence that PD is not associated with pervasive effects on lymphocyte proliferative activity. Finally, the inability of several studies to demonstrate differences in the production or levels of various cytokines in PD (e.g., Brambilla, Bellodi, & Perna, 1999; Rapaport & Stein, 1994; Weizman, Laor, Wiener, Wolmer, & Bessler, 1999), while limited in scope, further suggest that lymphocyte functional responses are not substantially altered in this disorder. Depressive and anxiety disorders often co-exist within individuals and families (Kessler et al., 1998; Weissman et al., 1993) and there is evidence for an interaction between depression and anxiety disorders in relation to immune activity in MD patients (Andreoli et al., 1992). The present study, in an effort to assess the immune system in PD, excluded subjects who met criteria for current MD. However, more than half of the PD subjects were found to have a history of prior MD. While several studies suggest that immune changes in MD are state dependent (Irwin, Lacher, & Caldwell, 1992; Ravindran, Griffiths, Merali, & Anisman, 1995; Schleifer, Keller, & Bartlett, 1999), the possibility that MD, as a ‘‘trait’’ or biological diathesis, may be associated with immune change warrants consideration. Hierarchical regression analyses were therefore conducted on an exploratory basis to assess the role of a history of depression in the associations found between PD
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and both lower B cell numbers and PHA responses. After first controlling (a priori) for age, gender, and ethnicity, MD history (present vs absent) was entered into the model, followed by PD (present vs absent). These analyses revealed no contribution of past MD to B cell levels, with the PD effect persisting after controlling for MD (p < :02, data not shown). For PHA response, however, there was evidence for an effect of past MD on each of the two higher doses of PHA (p < :1) and an associated loss of the PD effect for PHA (p > :5; data not shown). These preliminary observations suggest that effects on mitogen response in PD may be related to comorbid depressive disorder, even if not currently active. In contrast, PD appears to be associated independently with decreased B cells. NKCA, among the most consistently altered immune measures in patients with MD (Herbert & Cohen, 1993a), also did not differ in the present study between PD subjects and controls. This is consistent with the report of Koh and Lee (1998). One prior study (McDaniel, Risby, Stipetic, Jewart, & Caudle, 1994) did find altered (increased) NKCA in PD, although subjects in that study appeared to have substantial depressive symptoms (mean HDRS ¼ 18.1, although not meeting MD diagnostic criteria) in contrast to the current sample (HDRS ¼ 7.6). In consideration of that report, and as a first step toward assessing possible depression-PD interactions for NKCA, we conducted an hierarchical regression on NKCA controlling for past MD (and age, gender, and ethnicity), and still found no evidence for NKCA changes in PD after controlling for depression (data not shown). Studies are needed to explore NKCA as well as other immune measures in subgroups of PD patients defined by their levels of depressive symptoms and syndromes. The current findings must be considered in relation to the limitations of the study sample, and generalizations should be undertaken with caution. The sample included young to middle age adults; older adults with PD, as with MD (Schleifer, Keller, Bond, Cohen, & Stein, 1989), may show immune system changes not seen in younger PD adults. Gender differences may be important and effects specific to males would not have been readily detected from the small number of males in the sample. While the PD subjects and controls were rigorously matched and studied concurrently, the resulting sample was small and further studies with larger samples will be required to assess differences in assays such as PHA response.
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A number of substances to which patients with psychiatric disorders may have increased exposure could potentially influence immune function and confound assessment of links between the disorder itself and the immune system. Persons with current or recent exposure to substances of abuse or to psychotropic agents, especially those utilized for the treatment of panic disorder, were not included in the sample. Subjects who reported taking occasional benzodiazepines (n ¼ 3) were not excluded, however. To confirm that this limited use had not influenced the observed immune differences (i.e., on B cells and PHA), covariance analyses were conducted. No evidence for an effect of benzodiazepine use on the immune differences was found (data not shown). Similar exploratory analyses found no evidence that caffeine or alcohol use influenced the immune differences (data not shown). The current PD sample reported higher tobacco use than their matched controls and smoking has been associated with altered B cells and other immune measures (Tanigawa et al., 1998; Bijl, Horst, Limburg, & Kallenberg, 2001), effects that may be especially pronounced among psychiatric patients (Jung & Irwin, 1999). To determine whether smoking accounted for the decreased circulating B cells and possible mitogen effects in the PD subjects, we undertook exploratory covariance analyses controlling for cigarette use (data not shown). These revealed a significant positive association between smoking during the preceding two weeks and circulating B cells (p < :01) (data not shown), consistent with prior reports. When controlling for this effect in the covariance analysis, however, the association between PD and lower B cells was not diminished, but rather enhanced (p 6 :01). Increased smoking among patients with PD may mitigate the decreased circulating B cells in those persons, an effect that requires further investigation. Current and previous studies of patients with PD have shown only modest evidence for persistent immune system changes. These contrast with the more extensive and consistently described changes in immunity found in persons with major depression (MD) (Herbert & Cohen, 1993a). Whether the limited immune changes in PD are characteristic of the anxiety disorders as a group requires systematic investigation. Studies of both generalized anxiety disorder (GAD) (La Via et al., 1996) and posttraumatic stress disorder (PTSD) (Laudenslager et al., 1998) did not find altered circulating B cells, however, in one study
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(Koh & Lee, 1998), PHA mitogen response was found to be decreased in GAD as well as in PD patients. Future studies of immunity in psychiatric disorders will need to investigate the range of clinical symptomatology as well as of diagnostic specificity in assessing the association of anxiety and depression with immune system changes (see Andreoli et al., 1992). Among the factors to be considered along these clinical and diagnostic dimensions is the clinical symptomatology at the time of immune study. Patients meeting diagnostic criteria for PD, in contrast to most patients with anxiety or affective disorders, are likely to be studied during intervals in which they are not suffering with acute panic attacks, which tend to be episodic. It is therefore not known whether transient alterations occur in the immune system as a consequence of the panic episode itself, during which physiologic changes occur that share features with classical stress responses (Gorman et al., 2000). The absence of substantial and persistent immune system changes in PD patients during the interval between such episodes, however, suggests that altered immunity is not likely to have major health consequences in otherwise healthy young adults with this disorder. The limited immune system changes in PD, when contrasted with the broader effects reported in depressive disorders, provides further evidence for the specificity of immune changes among the psychiatric disorders.
Acknowledgments Supported in part by grants from the Upjohn Company, the New Jersey Medical School Biomedical Research Support Grant, and the Chernow Foundation. We thank Barbara ScottPlotner, Paula Lourenco, Karen Pearlman, Hong Lin Niu, Virginia Wasserman, Haftan Eckholdt, John Martinez, and Dr. Sally Czaja for their expert assistance in subject recruitment, data collection, and statistical support. Aspects of this work were presented in part at the annual meetings of the Society of Biological Psychiatry (1990) and the American Psychiatric Association (1998).
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Panic disorder and immunity: Few effects on circulating lymphocytes, mitogen response, and NK cell activity Steven J. Schleifer,* Steven E. Keller, and Jacqueline A. Bartlett Department of Psychiatry, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA Received 24 July 2001
Abstract Altered immune measures are commonly found in major depression (MD), however, less is known about the immune system in anxiety disorders. We examined quantitative and functional in vitro immune measures in patients with panic disorder (PD), which is often comorbid with MD. Fourteen otherwise healthy medication-free adults (ages 23–49; 11 female) meeting SCID-UP DSM-IIIR criteria for PD with agoraphobia and without current MD, were compared with 14 subjects free of PD, MD, or other major psychiatric disorders, matched by gender, age, and racial background. PD was associated with decreased percentage (p < :03) and total (p < :03) circulating CD19+ B lymphocytes, but no differences in other enumerative lymphocyte measures. Mitogen responses (Con A, PHA, PWM) did not differ except for possibly decreased PHA in PD (p < :06). NK cell activity did not differ between PD and control subjects. The few immune measure changes in PD contrast with those found in MD, providing further evidence for the specificity of immune changes in psychiatric disorders. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Panic disorder; Immunity; Lymphocytes; Mitogens; Phytohemagglutinin; NK cells; B cells; Anxiety disorders; Agoraphobia
1. Introduction An association between depressive disorders and altered immunity has been described in many studies (Herbert & Cohen, 1993a). Less is known about the immune system in panic disorder (PD), which shares some biological, clinical, and therapeutic characteristics with affective disorders, is often comorbid with major depression (MD), but appears to be a separate entity (Abelson & Curtis,
*
Corresponding author. Fax: 1-973-972-8305. E-mail address: [email protected] (S.J. Schleifer).
1996; Ballenger, 1999; Gorman, Kent, Sullivan, & Coplan, 2000; Kessler et al., 1998; Weissman et al., 1993). The possible isomorphism of panic disorder and increased stress reactivity (Gorman et al., 2000) further suggests the possibility of immune system changes in PD such as those regularly found following stress exposure (Herbert & Cohen, 1993b). Studies from our group found that comorbid anxiety disorders in depressed patients were associated with immune measures that differed from those found with MD alone (Andreoli et al., 1992). Studies of immunity in PD without MD have been limited and have tended to focus on a single
0889-1591/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 8 8 9 - 1 5 9 1 ( 0 2 ) 0 0 0 2 2 - 3
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immune dimension. To investigate whether persons with PD show immune changes comparable to those found in depressive disorders, we compared medication-free patients without medical disorders who met criteria for PD and not MD with matched subjects without a history of panic or depressive disorder. PD and non-PD subjects were compared with respect to the number and distribution of circulating leukocytes, lymphocytes and lymphocyte subsets, mitogen induced lymphocyte proliferation, and natural killer (NK) cell activity.
2. Method The study was approved by the Institutional Review Board of UMDNJ-New Jersey Medical School. Informed consent was obtained from all subjects. Eighteen subjects and 18 matched controls over 18 years of age were recruited, of whom 14 pairs were retained in the current analyses (four PD patients were found on further evaluation to meet criteria for current MD or medical exclusionary criteria). The PD subjects were recruited primarily from the outpatient Panic Disorder and Phobia Clinic at Hackensack University Medical Center, and from other clinical programs of the UMDNJ-New Jersey Medical School, Newark, NJ. A medical history, review of systems and vital signs were obtained along with standard laboratory screening. Potential subjects were excluded if they had evidence for diseases likely to have substantial effects on immunity (e.g., neoplastic, endocrine or immune disorders, acute infectious processes) or if they were taking medications with known immunologic effects. No subjects were taking psychotropic medications at study entry (and for at least several weeks beforehand) with the exception of three who were taking diazepam or lorazepam on an as needed basis. PD subjects were each studied on the same day as their matched control, who met the same medical inclusion/ exclusion criteria and had no history of MD, PD, or other major psychiatric disorders. Control subjects were matched for age, sex, and race and were recruited from UMDNJ and Hackensack University Medical Center personnel and from surrounding communities. Diagnostic assessments were made utilizing the SCID-UP, a semi-structured diagnostic instrument designed to generate DSMIII-R psychiatric diagnoses, and modified to include more extensive data in relation to panic disorder (Spitzer,
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Williams, & Gibbon, 1986; American Psychiatric Association, 1987). The Hamilton anxiety scale (HARS) (Hamilton, 1959) and the Hamilton depression rating scale (HDRS) (Hamilton, 1967) were utilized. All assessments were made by trained interviewers, who had achieved interrater reliability (>0.80) for DSM-IIIR diagnoses (Kappa) and for the HAS and HDRS (intraclass correlation coefficient). Upon completion of each interview, 30 ml of blood were collected for immune studies. Control subjects were compensated for their time, effort, and expenses. Subjects were studied, in general, during the late morning or early afternoon. All data were collected with interviewers and laboratory technicians ‘‘blinded’’ to the maximum extent possible: Technicians received blood samples with only a sequential number for identification and interviewers were blind to the biological data. Immune assays were carried out the same day as venipuncture. Blood samples were collected into a heparinized syringe (preservative-free heparin). Total white blood cell and differential counts were performed by standard techniques. Lymphocytes were separated from whole blood by centrifugation on a Ficoll–Hypaque gradient (Pharmacia Fine Chemicals, Piscataway, NJ). Aliquots of these lymphocytes were utilized in the in vitro assays. Lymphocyte subsets [T (CD3+), B (CD19+), NK (CD56+), T4 (CD4+), T8 (CD8+), T4 inducer of help (CD29+), T4 inducer of suppression/naive cells (CD45RA+), and activated T cells (HLADR+)] were assessed by flow cytometry using a battery of paired (FITC and PE) monoclonal antisera (Coulter Cytometry, Hialeah, FL). The proliferative response of isolated peripheral blood lymphocytes to mitogens was assessed according to standard techniques (Keller et al., 1981). Two peak doses were utilized for phytohemagglutinin (PHA; Wellcome Reagents, Beckenham, UK), Concanavalin A (Con A; Calbiochem, San Diego, CA), and pokeweed mitogen (PWM; GIBCO, Gaithersburg, MD). Lymphocyte stimulation data are expressed as counts per minute (cpm) in the stimulated cultures minus the cpm in the unstimulated cultures (D cpm). D cpm data were log transformed for all analyses to stabilize the variance. NK cell activity (NKCA) was assessed utilizing K562 target cells according to standard methods as modified by the investigators (Georgescu & Keller, 1987). Analysis of variance (ANOVA) for repeated measures was used to test overall mean differences between the PD and control groups for the
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mitogen assays (mitogen dose as the repeated measure) and NK cell function (effector:target cell ratio as the repeated measure) and paired t tests were used to test for differences in the other immune variables. For exploratory analyses assessing the contribution of potential modulating variables, covariance analyses, and hierarchical multiple regression analyses were used.
3. Results The characteristics of the PD and matched non-PD subjects are shown in Table 1. The age range for the PD subjects was 23–49. The female/ male ratio of 3:1 is in keeping with the higher prevalence of PD in women. HARS scores ranged from 9 to 23. HDRS scores (1–23) ranged from levels associated with none to moderate affective
Table 1 Subject characteristics: Means SD (range) Variable
PD subjects Matched (n ¼ 14) controls (n ¼ 14)
Age
37:5 8:2 (23–49)
37:2 7:8 (24–51)
Gender Male Female
3 11
3 11
Race African–American Caucasian
1 13
1 13
Hamilton depression scale 7:6 5:7 (1–23) (HDRS)
0:9 1:2 (0–4)
Past MDD Yes No
9 5
0 14
Tobacco users Packs/day past 2 years Packs/day past 2 weeks
8 0:54 0:75 0:36 0:63
7 0:04 0:13 0:07 0:27
Hamilton anxiety scale (HARS)
14:0 4:1 (9–23)
1:3 2:4 (0–8)
Alcohol: Drinks/day Past 2 years Past 24 h
3:1 3:5 0:5 1:4
1:8 2:3 0:7 1:5
1:9 1:6
2:7 1:9
Caffeinated beverages past 24 h p < :01. ** p < :05. *
symptoms. Nine of the 14 PD subjects met criteria for prior major depression. PD patients reported significantly more tobacco use, but did not differ significantly in the use of alcohol or caffeine. All PD subjects met criteria for agoraphobia. 3.1. Enumerative measures As seen in Table 2, comparisons of the percentages of leukocytes, lymphocytes, and lymphocyte subsets for the PD patients and their matched controls showed decreased circulating percentages of (CD19+) B cells (p < :025). There were no other differences between the PD patients and their matched controls in the distribution of circulating leukocytes and lymphocyte subsets. Comparison of the number of circulating leukocytes and lymphocytes for the PD patients and their matched controls (Table 3) revealed, similarly, decreased circulating B cells (p < :03) and no differences in the other circulating leukocytes, lymphocytes, or lymphocyte subsets. 3.2. Functional measures As seen in Fig. 1, lymphocyte stimulation responses for the mitogens Con A, PHA, and PWM appeared to be comparable for patients with PD and their matched controls. Repeated measures ANOVA revealed no significant differences for any of the mitogens, except for a possibly decreased overall response to PHA in the PD subjects: Con A (group: F ¼ 0:36; df ¼ 1; 11, p n.s.; dose: F ¼ 15:06; df ¼ 1; 11; p < :005; group X dose: F ¼ 0:07; df ¼ 1; 11, p n.s.); PHA (group: F ¼ 4:91; df ¼ 1; 7; p < :06; dose: F ¼ 34:61; df ¼ 1; 7; p < :001; group X dose: F ¼ 0:16; df ¼ 1; 7, p n.s.); PWM: (group: F ¼ 1:67; df ¼ 1; 9; p n.s.; dose: F ¼ 0:53; df ¼ 1; 9, p n.s.; group X dose: F ¼ 0:39; df ¼ 1; 9, p n.s.). As seen in Fig. 2, NKCA was comparable for PD subjects and their matched controls. ANOVA for repeated measures on NKCA revealed no significant PD or PD by effector:target ratio effects (group: F ¼ 0:01; df ¼ 1; 11, p n.s.; E:T ratio: F ¼ 81:44; df ¼ 2; 10; p < :0001; group X E:T ratio: F ¼ 0:94; df ¼ 2; 10, p n.s.).
4. Discussion The present study of otherwise healthy, medication free patients meeting criteria for PD with agoraphobia, but not current MD, showed no
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Table 2 Peripheral leukocyte measures: Panic disorder subjects and matched controls (cells as percent of leukocytes/lymphocytes SD) Variable
Panic disorder subjects
Matched control subjects
t
Paired n
p
Lymphocytes Granulocytes Monocytes T lymphocytes B lymphocytes CD4+ lymphocytes CD8+ lymphocytes CD29+ lymphocytes (inducers of help) CD45RA+ lymphocytes (inducers of suppression) CD56+ lymphocytes (NK cells) HLA-DR+ lymphocytes (activated T cells)
31:2 8:3 61:1 8:2 7:17 2:42 83:4 4:6 10:6 3:5 50:9 8:3 21:9 4:0 23:2 5:9 22:9 8:9 5:08 4:50 4:70 2:12
36:2 5:8 57:3 5:9 6:03 0:82 81:7 4:2 13:9 3:9 50:2 6:9 22:5 5:1 23:3 4:5 21:2 5:6 3:88 2:13 5:46 4:15
1:58 1.28 1.62 1.38 2:58 0.32 0:38 0:08 0.77 0.80 0:76
11 11 11 14 14 14 14 8 9 14 14
n.s. n.s. n.s. n.s. <.025 n.s. n.s. n.s. n.s. n.s. n.s.
Table 3 Peripheral leukocyte measures: Panic disorder subjects and matched controls (mean cells/ml 106 SD) Variable
Panic disorder subjects
Matched control subjects
t
Paired n
p
Leukocytes Lymphocytes Granulocytes Monocytes T lymphocytes B lymphocytes CD4+ lymphocytes CD8+ lymphocytes CD29+ lymphocytes (inducers of help) CD45RA+ lymphocytes (inducers of suppression) CD56+ lymphocytes (NK cells) HLA-DR+ lymphocytes (activated T cells)
7:20 2:60 2:33 0:72 4:81 1:94 0:53 0:16 1:96 0:63 0:25 0:11 1:20 0:47 0:50 0:19 0:52 0:14 0:59 0:37 0:10 0:11 0:11 0:05
7:19 1:72 2:70 0:97 4:16 1:08 0:44 0:16 2:24 0:82 0:36 0:13 1:35 0:46 0:60 0:33 0:66 0:20 0:62 0:31 0:11 0:07 0:14 0:08
0.01 1:01 1.12 1.31 0:91 2:55 0:80 0:94 1:59 0:27 0:22 0:98
14 11 11 11 11 11 11 11 8 9 11 11
n.s. n.s. n.s. n.s. n.s. <:03 n.s. n.s. n.s. n.s. n.s. n.s.
evidence for significant differences from matched controls in the number or distribution of leukocytes, lymphocytes, or lymphocyte subsets, lymphocyte proliferative responses to T or B cell mitogens, or NK cell activity. The exceptions were a decrease in the proportion and number of CD19+ cells (B lymphocytes) and a possible decrease in PHA response. Other studies have investigated the proportion of CD19+ cells in PD, including two that found no differences from controls (Marazziti et al., 1992; Manfro et al., 2000) and two that found increased CD19+ cells in PD patients (Perini et al., 1995; Rapaport, 1998). These reports included study samples that appear to have been demographically and clinically similar to the present sample, with the exception of the small proportion of males in the current study and our only including PD
subjects with agoraphobia. Future studies are needed to clarify’ factors such as gender and disorder subtype and tobacco and other substance use (see below) that may interact in the apparent association between PD and circulating B cells. Studies of the distribution of lymphocytes and other lymphocyte subsets in PD have, in general, not replicated several single reports of differences from non-PD subjects. Single studies have reported decreased circulating total lymphocytes (Ramesh, Yeragani, Balon, & Pohl, 1991), decreased CD3+ (total T) cells (Perini et al., 1995), and decreased CD4+ (T helper) cells (Marazziti et al., 1992). For each of these observations, at least one other study (e.g., Manfro et al., 2000; Rapaport, 1998) found no significant differences between PD patients and controls, and the current study similarly found no differences in these
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Fig. 1. Response to PHA, Con A, and Pokeweed Mitogen (log transformed; means SEM) for panic disorder subjects (PD) and matched controls (Ctrl).
Fig. 2. NK cell activity (% cytotoxicity) (means SEM) for panic disorder subjects (PD) and matched controls (Ctrl).
measures. One study found increased total leukocytes in PD (Ramesh et al., 1991) and one increased NK cells (Rapaport, 1998), findings that were also not confirmed in other studies (e.g., Manfro et al., 2000; Marazziti et al., 1992) including the present report. There are now two
reports of increased HLA-DR+ cells in PD (Perini et al., 1995; Rapaport, 1998), an effect that, as noted (Rapaport, 1998), may have been a consequence of the increased B cells found in the PD subjects rather than an indication of increased activated T cells. Correspondingly, both the current report and that of Marazitti et al. (1992), which did not find increased B cells, found no increase in HLA-DR+ cells. Mitogen responses, a measure of the functional capacity of T or B cells to proliferate in response to a nonspecific challenge, has been found to be altered in subgroups of patients with depressive disorders and following life stress (Herbert & Cohen, 1993a,b). The current findings, showing possibly decreased PHA in PD (p < :06), indicate a need for further investigation with larger samples considering two prior reports of no PHA differences (Brambilla et al., 1992; Surman et al., 1986) but one of decreased PHA response in PD (Koh & Lee, 1998). The failure to detect differences in PWM response are consistent with the one previous report in PD (Surman et al., 1986). The lack of differences for Con A, which apparently has not been assessed previously, provides additional evidence that PD is not associated with pervasive effects on lymphocyte proliferative activity. Finally, the inability of several studies to demonstrate differences in the production or levels of various cytokines in PD (e.g., Brambilla, Bellodi, & Perna, 1999; Rapaport & Stein, 1994; Weizman, Laor, Wiener, Wolmer, & Bessler, 1999), while limited in scope, further suggest that lymphocyte functional responses are not substantially altered in this disorder. Depressive and anxiety disorders often co-exist within individuals and families (Kessler et al., 1998; Weissman et al., 1993) and there is evidence for an interaction between depression and anxiety disorders in relation to immune activity in MD patients (Andreoli et al., 1992). The present study, in an effort to assess the immune system in PD, excluded subjects who met criteria for current MD. However, more than half of the PD subjects were found to have a history of prior MD. While several studies suggest that immune changes in MD are state dependent (Irwin, Lacher, & Caldwell, 1992; Ravindran, Griffiths, Merali, & Anisman, 1995; Schleifer, Keller, & Bartlett, 1999), the possibility that MD, as a ‘‘trait’’ or biological diathesis, may be associated with immune change warrants consideration. Hierarchical regression analyses were therefore conducted on an exploratory basis to assess the role of a history of depression in the associations found between PD
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and both lower B cell numbers and PHA responses. After first controlling (a priori) for age, gender, and ethnicity, MD history (present vs absent) was entered into the model, followed by PD (present vs absent). These analyses revealed no contribution of past MD to B cell levels, with the PD effect persisting after controlling for MD (p < :02, data not shown). For PHA response, however, there was evidence for an effect of past MD on each of the two higher doses of PHA (p < :1) and an associated loss of the PD effect for PHA (p > :5; data not shown). These preliminary observations suggest that effects on mitogen response in PD may be related to comorbid depressive disorder, even if not currently active. In contrast, PD appears to be associated independently with decreased B cells. NKCA, among the most consistently altered immune measures in patients with MD (Herbert & Cohen, 1993a), also did not differ in the present study between PD subjects and controls. This is consistent with the report of Koh and Lee (1998). One prior study (McDaniel, Risby, Stipetic, Jewart, & Caudle, 1994) did find altered (increased) NKCA in PD, although subjects in that study appeared to have substantial depressive symptoms (mean HDRS ¼ 18.1, although not meeting MD diagnostic criteria) in contrast to the current sample (HDRS ¼ 7.6). In consideration of that report, and as a first step toward assessing possible depression-PD interactions for NKCA, we conducted an hierarchical regression on NKCA controlling for past MD (and age, gender, and ethnicity), and still found no evidence for NKCA changes in PD after controlling for depression (data not shown). Studies are needed to explore NKCA as well as other immune measures in subgroups of PD patients defined by their levels of depressive symptoms and syndromes. The current findings must be considered in relation to the limitations of the study sample, and generalizations should be undertaken with caution. The sample included young to middle age adults; older adults with PD, as with MD (Schleifer, Keller, Bond, Cohen, & Stein, 1989), may show immune system changes not seen in younger PD adults. Gender differences may be important and effects specific to males would not have been readily detected from the small number of males in the sample. While the PD subjects and controls were rigorously matched and studied concurrently, the resulting sample was small and further studies with larger samples will be required to assess differences in assays such as PHA response.
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A number of substances to which patients with psychiatric disorders may have increased exposure could potentially influence immune function and confound assessment of links between the disorder itself and the immune system. Persons with current or recent exposure to substances of abuse or to psychotropic agents, especially those utilized for the treatment of panic disorder, were not included in the sample. Subjects who reported taking occasional benzodiazepines (n ¼ 3) were not excluded, however. To confirm that this limited use had not influenced the observed immune differences (i.e., on B cells and PHA), covariance analyses were conducted. No evidence for an effect of benzodiazepine use on the immune differences was found (data not shown). Similar exploratory analyses found no evidence that caffeine or alcohol use influenced the immune differences (data not shown). The current PD sample reported higher tobacco use than their matched controls and smoking has been associated with altered B cells and other immune measures (Tanigawa et al., 1998; Bijl, Horst, Limburg, & Kallenberg, 2001), effects that may be especially pronounced among psychiatric patients (Jung & Irwin, 1999). To determine whether smoking accounted for the decreased circulating B cells and possible mitogen effects in the PD subjects, we undertook exploratory covariance analyses controlling for cigarette use (data not shown). These revealed a significant positive association between smoking during the preceding two weeks and circulating B cells (p < :01) (data not shown), consistent with prior reports. When controlling for this effect in the covariance analysis, however, the association between PD and lower B cells was not diminished, but rather enhanced (p 6 :01). Increased smoking among patients with PD may mitigate the decreased circulating B cells in those persons, an effect that requires further investigation. Current and previous studies of patients with PD have shown only modest evidence for persistent immune system changes. These contrast with the more extensive and consistently described changes in immunity found in persons with major depression (MD) (Herbert & Cohen, 1993a). Whether the limited immune changes in PD are characteristic of the anxiety disorders as a group requires systematic investigation. Studies of both generalized anxiety disorder (GAD) (La Via et al., 1996) and posttraumatic stress disorder (PTSD) (Laudenslager et al., 1998) did not find altered circulating B cells, however, in one study
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(Koh & Lee, 1998), PHA mitogen response was found to be decreased in GAD as well as in PD patients. Future studies of immunity in psychiatric disorders will need to investigate the range of clinical symptomatology as well as of diagnostic specificity in assessing the association of anxiety and depression with immune system changes (see Andreoli et al., 1992). Among the factors to be considered along these clinical and diagnostic dimensions is the clinical symptomatology at the time of immune study. Patients meeting diagnostic criteria for PD, in contrast to most patients with anxiety or affective disorders, are likely to be studied during intervals in which they are not suffering with acute panic attacks, which tend to be episodic. It is therefore not known whether transient alterations occur in the immune system as a consequence of the panic episode itself, during which physiologic changes occur that share features with classical stress responses (Gorman et al., 2000). The absence of substantial and persistent immune system changes in PD patients during the interval between such episodes, however, suggests that altered immunity is not likely to have major health consequences in otherwise healthy young adults with this disorder. The limited immune system changes in PD, when contrasted with the broader effects reported in depressive disorders, provides further evidence for the specificity of immune changes among the psychiatric disorders.
Acknowledgments Supported in part by grants from the Upjohn Company, the New Jersey Medical School Biomedical Research Support Grant, and the Chernow Foundation. We thank Barbara ScottPlotner, Paula Lourenco, Karen Pearlman, Hong Lin Niu, Virginia Wasserman, Haftan Eckholdt, John Martinez, and Dr. Sally Czaja for their expert assistance in subject recruitment, data collection, and statistical support. Aspects of this work were presented in part at the annual meetings of the Society of Biological Psychiatry (1990) and the American Psychiatric Association (1998).
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