Twenty six subjects (eleven women and fifteen men) who were medically healthy, as determined by medical history, physical examination, and laboratory testing, were recruited between October 2006 and June 2008. All subjects fulfilled criteria for Never Mentally Ill as determined by Structured Clinical Interview for Diagnostic and Statistical Manual - IV (SCID). Females and males did not differ in mean ± SD age (37.2 ± 9.6 vs. 36.3 ± 10.4 years; t = 0.2, P = 0.82), education level (15.9 ± 1.8 vs. 15.3 ± 1.8; t = 0.9, P = 0.37) mean body mass index [BMI] (23.8 ± 3.8 vs. 24.9 ± 3.7; t = 0.7, P = 0.47), or ethnicity (nonwhite: 36.4% vs. 20.0%; χ2 = 0.86, P = 0.35) 8=26.9 [SD=3.6]; 35.7% were non-white). Without restricting assessments to a single point in the menstrual cycle, females and males did not differ in mean levels of progesterone (1.2 ± 0.6 ng/ml vs. 1.0 ± 0.6; t = 1.1, P = 0.30) or estradiol (67.1 ± 62.3 ng/ml vs. 37.5 ± 9.7 ng/ml; t = 1.9, P = 0.08). Past or current Axis I DSM-IV psychiatric disorder, use of psychotropic medication, regular use of nonsteroidal anti-inflammatory medications, and tobacco smoking were exclusionary. Subjects regularly slept between 22:30 and 7:30 h as confirmed by 2-week sleep diaries. The study was approved by the UCLA Institutional Review Board.
Subjects participated in a PSD protocol that was conducted on the UCLA General Clinical Research Center (GCRC) as previously described (Irwin et al., 2006
). The PSD night immediately followed the baseline night and all subjects remained on the GCRC during the day with behavioral monitoring to prevent any napping behavior. Acquisition of blood samples occurred via an indwelling venous forearm catheter at 08:00, 12:00, 16:00, 20:00, and 23:00 h during baseline and after PSD. Hence, a total of 5 measures were obtained prior to sleep deprivation (i.e., baseline) with 5 additional measures obtained after PSD. Blood samples were held at room temperature between collection and assay, as we have found up to a 10 h- interval does not impact assay results; baseline and PSD samples were treated similarly in terms of hold times.
Monocyte intracellular production of IL-6 and TNF-α was assessed by flow cytometry using peridinin chlorophyll protein—labeled CD14 monoclonal antibody, allophycocyanin-labeled anti- TNF-a monoclonal antibody, and phycoerythrinlabeled IL-6 antibody. (Collado-Hidalgo et al., 2006
). Heparin-treated blood (1 mL) was mixed with 100 pg/mL LPS (Sigma, St. Louis, MO) and 10 Ag/mL brefeldin A (Sigma) and incubated for 4 hours at 37°C in a platform mixer followed by an overnight incubation at 4°C. RBCs were lysed in fluorescence-activated cell sorting lysing solution (BD Biosciences), remaining cells were permeabilized in fluorescence-activated cell sorting permeabilizing buffer (BD Biosciences), and fluorescence conjugatedantibodies were added for 30 minutes at room temperature in the dark. Cells were then washed and resuspended in 1% paraformaldehyde for assay on a Coulter Elite flow cytometer using the Coulter Elite software. Forward scatter and side scatter were used to gate on monocytes and granulocytes. About 12,000 CD14+ events were counted to determine the percentage of cytokine-secreting monocytes, with quadrant coordinates set based on unstimulated monocytes cells. To determine percentage of stimulated cells expressing TNF-α, IL-6, or co-expressing TNF-α and IL-6, unstimulated cytokine-positive event percentages were subtracted from stimulated percentages to obtain net stimulated cytokine positive event percentages. We have previously reported that PSD does not alter absolute numbers of monocytes (Irwin et al. 1996).
Data were analyzed using SAS version 9.13 for Windows. To determine the effects of PSD on monocyte intracellular proinflammatory cytokine expression in females and males, repeated measures mixed model ANOVA was performed using a 2 (sex: female, male) × 2 (condition: baseline, PSD) × 5 (time: 08:00, 12:00, 16:00, 20:00, 23:00 h) design, covarying for age, education, ethnicity, BMI, progesterone and estradiol. Analyses were conducted for monocytes expressing TNF-α, IL-6, or co-expressing TNF-α and IL-6, as well as the aggregate.