Participants

We analyzed 35 individuals with current PTSD (19 male, 16 female; mean age = 32.6, range 16–51) according to the DSM-IV [4] and 25 healthy control subjects (8 male, 17 female; mean age = 26.8 years, range 18–45). PTSD patients were refugees, with chronic (mean symptom duration = 7.6 years, SD = 4.6) and severe (mean sum score in the Clinician Administered PTSD Scale, CAPS [25] = 80.0, SD = 17.5) forms of PTSD due to multiple highly stressful war and torture experiences, mainly experienced during late adolescence and adulthood. In addition to the PTSD diagnosis, 27 patients met the DSM-IV criteria for a current major depressive episode. Thirteen PTSD patients reported current intake of hypnotic, anxiolytic, antidepressant or neuroleptic medication and one woman reported the use of oral contraceptives. Thirty-seven percent of the PTSD patients were smokers (for subjects’ characteristics see Table 1). All patients were recruited from the Psychotrauma Research and Outpatient Clinic for Refugees, University of Konstanz, Germany. The healthy control group was recruited through advertisement and was comparable to the patient group with regard to region of origin. Except for four women reporting the intake of oral contraceptives, all control subjects were free of medication. Sixteen percent of the control subjects were smokers (see Table 1).

Table 1 Sociodemographic and clinical characteristics of PTSD patients and controls Full size table

Exclusion criteria for the study were intake of glucocorticoids or acute (1 PTSD patient and 2 controls) and chronic (1 PTSD patient and 3 controls) somatic illnesses. In addition, control subjects were excluded if they met the criteria for any mental disorder according to DSM-IV (n = 4), or reported intake of psychotropic medication (n = 2). PTSD patients were excluded if they met the criteria for comorbid alcohol or substance abuse and dependence (n = 3) or a current or past history of a psychosis (n = 1) according to DSM-IV. Furthermore, participants were screened for possible HIV and hepatitis A, B and C infections. All samples were negative for HIV or hepatitis C. Subjects classified with acute or chronic hepatitis A or B (3 PTSD patients and 3 controls) were excluded from the study, reducing the initially enrolled sample of 44 individuals with PTSD and 39 controls to 35 PTSD patients and 25 control participants.

Basal plasma cytokine measurements were obtained from all subjects (PTSD, n = 35; healthy controls, n = 25). Spontaneous and LPS-induced cytokine production by cultured PBMCs was investigated in a subsample of 16 PTSD patients and 18 control subjects (see Table 2 for the respective subject characteristics).

Table 2 Sociodemographic and clinical characteristics of the subgroup of PTSD patients and control subjects for which we analyzed cytokine production by PBMCs Full size table

Procedure

All participants underwent an extensive standardized clinical interview administered by experienced clinical psychologists and trained translators, starting always at 10:00 a.m.: Upon arrival at the outpatient clinic, procedures were explained to the participants and written informed consent was obtained. Subsequently, blood (t 1 ) for white blood cell (WBC) differential counts, plasma cytokines, and PBMC isolation was drawn. Afterwards, sociodemographic information as well as medical information was acquired. Somatic symptoms were assessed using a shortened version of Screening for Somatoform Symptoms 7 SOMS-7, [26]. During the second part of the interview, participants were interviewed in a standardized manner about their individual traumatic experiences using the event checklist of the CAPS [25] and the vivo checklist of war, detention and torture events[27], which assesses common traumatic experiences in conflict regions and during torture. Subsequently, PTSD symptom frequency and severity were assessed with the CAPS [25]. Finally, the Mini International Neuropsychiatric Interview M.I.N.I., [28] was applied to screen for potential comorbid mental disorders. In addition, depressive symptoms were assessed with the Hamilton Depression Rating Scale HAM-D, [29]. One week after the standardized clinical interview the participants were invited for a second time. Again, blood samples for plasma cytokines were collected at 10:00 am (t 2 ), to test for the stability of basal (t 1 ) cytokine levels.

All procedures were approved by the Ethics Committee of the University of Konstanz and were carried out in accordance with the Declaration of Helsinki 2008.

WBC differential count

Absolute numbers of leukocytes, lymphocytes, neutrophils, monocytes, eosinophils, and basophils were obtained using an automated hematology analyzer (XT-2000i, Sysmex, Horgen, Switzerland).

Cell culture

Peripheral blood mononuclear cells (PBMCs) were isolated from citrated blood by density gradient centrifugation using commercially available cell preparation tubes (Vacutainer® CPT™ Cell Preparation Tube, BD Biosciences, Franklin Lakes, NY, USA) according to the manufacturer’s instructions. Freshly isolated PBMCs (2 × 105) were suspended in 200 μl RPMI 1640 containing 10% fetal bovine serum and were cultured for 24 h in 96-well flat-bottom culture plates in presence or absence of 1 μg/ml lipopolysaccharide (LPS) from Salmonella abortus equi (Sigma-Aldrich, Taufkirchen, Germany).

Cytokine analysis

Cytokine levels in plasma and culture supernatants were quantified using multiplex bead-based assays (Bio-Plex Cytokine Assays, Bio-Rad Laboratories, Hercules, CA, USA). Samples were prepared according to the manufacturer’s instructions and were analyzed in triplicate on a dual-laser flow cytometer equipped with high throughput sampler (LSR II, BD Immunocytometry Systems, San Jose, CA, USA). Absolute cytokine levels were calculated based on the mean fluorescence intensity of cytokine standards. Detection limits of the assays, defined as the mean of background value plus 3 SD, were 0.07 pg/ml (IL-6), 0.75 pg/ml (IL-8), 0.14 pg/ml (IL-10), 0.24 pg/ml (TNF-α), and 0.38 pg/ml (MCP-1), respectively.

Statistical analyses

Data analysis was performed using R 2.15.2 [30]. Group comparisons with respect to sociodemographic and clinical variables were performed using chi-square tests for categorical data and t-tests for continuous data. Basal plasma and LPS-stimulated cytokine levels were analyzed using ANOVAs with group (PTSD patients vs. controls) as independent variable. Since some of the immunological variables did not met requirements for parametric testing (normality of residuals), statistical significance for the immune measures was assessed by nonparametric permutation tests. In each case, the full model and a reduced model omitting the factor(s) of interest were fitted and the statistic of interest (usually an F statistic) was calculated. Next, residuals under the reduced model were randomly permuted 10,000 times. In each case, the randomly permuted residuals were added back to the (non-permuted) fitted values under the reduced model. The resulting randomized dependent values were then again used in fitting full and reduced models, yielding a “permutation” statistic. The p values reported below are given by the position of the original statistic in the empirical distribution of the permutation statistic [31, 32]. In each case, we also investigated gender as well as smoking status as a covariate. We also wanted to control for the age difference between groups. However, as both groups differed significantly in age, the preconditions for calculating and interpreting an Analysis of Covariance were not fulfilled [33, 34]. In order to nevertheless understand our data better, we calculated Spearman correlations between age and dependent variables separately for the PTSD and control group.

Reported p values represent exact p values. In addition, we report p values corrected for multiple comparisons with Holm’s stepwise procedure [35], applied first for the five cytokines measured in plasma and then for the three cytokines produced by PBMCs measured in presence or absence of LPS. Correlations were computed using Spearman’s rank correlation coefficient.