Study groups

This study was approved by the University of Illinois at Chicago, College of Medicine Institutional Review Board and all participants consented to participate in this study. The study was performed in a blinded manner. A total of 110 fibromyalgia patients and 91 control subjects were recruited. Patient characteristics are shown in Table 1. All patients had fibromyalgia symptoms and had been diagnosed with fibromyalgia for at least one year. Patients underwent two separate and independent physical examinations to confirm that they met the criteria according to the current standards of the American College of Rheumatology [1]. All fibromyalgia patients had been off FDA-approved fibromyalgia drugs (milnacipran, pregabalin, duloxetine) for at least two weeks. Patients with any other potentially confounding variables were excluded, i.e., if they had any other rheumatologic disorders, any autoimmune or immunologic diseases, any inflammatory disorder, any infectious disorder or any neoplastic disorder or were receiving anti-cancer treatment or had been taking medications affecting the immune system, including any anti-inflammatory medications. Matched control subjects lacked a history of any type of chronic or acute illnesses and none were using any medications, OTC or prescription drugs that had any known immunologic effects. They ranged in age from 18 years to 76 years. The median age was 39 years, and the average age was 40 years. Of the 91 control subjects, 87 were Caucasian.

Table 1 Characteristics of patients Full size table

Isolation of PBMC

Twenty eight (28) mL of blood were obtained from patients and healthy volunteers after obtaining their consent for research. Blood was withdrawn in four 7 mL tubes containing 0.081 mL of 15% K 3 EDTA solution (BD Vacutainer®, BD, Franklin Lakes, NJ) by venipuncture. The samples were coded and shipped to the processing lab in insulated containers at room temperature. Once received, the blood was transferred to a 50ml tube, mixed, split in two 50 ml tubes and diluted 1:1 in GIBCO® Hank’s balanced salt solution (HBSS, Invitrogen, Carlsbad, CA). The resulting suspension was layered on top of 15 ml Ficoll (Histopaque®-1077, Sigma-Aldrich, St. Louis, MO) in 50 mL tubes and centrifuged at 700g for 20 minutes. The layer containing PMBC was harvested and transferred into a new 50 mL tube. The tube was filled with HBSS and the contents were mixed by gentle rocking. Cells were collected by centrifugation at 850g for 10 minutes and resuspended in 10 mL complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin mixture and 1% L-glutamine solution (Invitrogen, Carlsbad, CA).

PBMC culture

Cells were suspended at a concentration of 10 [6] cells/mL in a supplemented complete RPMI medium. A 3 mL suspension was placed in six-well tissue culture plates (Nalge Nunc International, Rochester, NY) and either 10 μg/mL PHA-P (Sigma-Aldrich®, St. Louis, MO) or a mixture of 1 μg/mL phorbol-12-myristate-13-acetate (PMA) and 1 μg/mL ionomycin (EMD Biosciences, Inc, Darmstadt, Germany) was added to the wells in triplicate. No chemicals were added to three control plates which were used to determine the basal levels of expression. Plates were incubated in a CO 2 water jacketed incubator (Forma Scientific, Marjetta, OH) for 18 hours.

Cytokine assay

After overnight culture of PBMC in medium alone or in the presence of mitogens, 1 mL supernatant was removed from each plate, centrifuged at 16,000g at +4°C, for 10 minutes and clear cell-free culture samples were collected, placed in a new 1.7 mL tube and frozen at -80°C. Cytokine and chemokine concentrations in plasma as well as in culture supernatants were determined using a multiplex immunoassay based on the Luminex xMAP™ bead array technology. A custom panel of antibody-conjugated beads for measuring eight human cytokines (IL-5, IL-6, IL-8, IFN-γ, IL-10, MIP-1α, MIP-1β, MCP-1) (BioRad Laboratories, Hercules, CA) was used in the assay according to the manufacturer's instructions. Cytokine concentrations were determined in both undiluted and diluted samples. Blank medium was used as a negative control. Serial dilutions of cytokine standards were run in duplicates in each assay; their readings were used for calculating standard curves. In addition, pooled culture supernatants obtained from activated cells served as a positive control. Fluorescence was measured with a Bio-Plex 200 fluorescence bead reader (BioRad Laboratories, Hercules, CA).

Statistical analysis

Fluorescence intensities were transferred into R [19] for converting to concentration values. The standard curves were fitted using the 4-parameter logistic (4-PL) or, in cases when the 4-PL model failed, the 5-parameter logistic (5-PL) model [20]. In total, 192 (24 plates times 8 cytokines) standard curves were fitted. The concentrations of cytokines in the plasma of controls and FM patients were compared using an unpaired two-sided t-test with unequal variance. This same test was used to compare the concentrations of cytokines in unstimulated and stimulated PBMC cultures of controls. The variances and means of the cytokine concentrations in patients and healthy individuals were compared using the F-test and t-test, respectively. The differences in variance were not detected at α=0.1, except for IL-5 in the PHA challenge. The descriptive statistics of the groups as well as the P-values of the latter t-test were also calculated. In order to determine whether the cytokine concentrations after PHA or PMA stimulations changed in the same direction for each individual patient, we calculated the pair-wise correlations between the cytokines using Spearman's rank correlation test. For all of the statistical tests, we used the stats package for R [19].