Cytokine and chemokine profiles in fibromyalgia, rheumatoid arthritis and systemic lupus erythematosus: a potentially useful tool in differential diagnosis
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- Wallace, D.J., Gavin, I.M., Karpenko, O. et al. Rheumatol Int (2015) 35: 991. doi:10.1007/s00296-014-3172-2
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Making a correct diagnosis is pivotal in the practice of clinical rheumatology. Occasionally, the consultation fails to provide desired clarity in making labeling an individual as having fibromyalgia (FM), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). A chemokine and cytokine multiplex assay was developed and tested with the goal of improving and achieving an accurate differential diagnosis. 160 patients with FM, 98 with RA and 100 with SLE fulfilling accepted criteria were recruited and compared to 119 controls. Supernatant cytokine concentrations for IL-6, IL-8, MIP-1 alpha and MIP-1 beta were determined using the Luminex multiplex immunoassay bead array technology after mitogenic stimulation of cultured peripheral blood mononuclear cells. Each patient’s profile was scored using a logistical regression model to achieve statistically determined weighting for each chemokine and cytokine. Among the 477 patients evaluated, the mean scores for FM (1.7 ± 1.2; 1.52–1.89), controls (−3.56 ± 5.7; −4.59 to −2.54), RA (−0.68 ± 2.26; −1.12 to −0.23) and SLE (−1.45 ± 3.34, −2.1 to −0.79). Ninety-three percent with FM scored positive compared to only 11 % of healthy controls, 69 % RA or 71 % SLE patients had negative scores. The sensitivity, specificity, positive predictive and negative predictive value for having FM compared to controls was 93, 89, 92 and 91 %, respectively (p < 2.2 × 10−16). Evaluating cytokine and chemokine profiles in stimulated cells reveals patterns that are uniquely present in patients with FM. This assay can be a useful tool in assisting clinicians in differentiating systemic inflammatory autoimmune processes from FM and its related syndromes and healthy individuals.
KeywordsFibromyalgia Rheumatoid arthritis Systemic lupus erythematosus Cytokines Chemokines Peripheral blood mononuclear cells Phytohemagglutinin
The differential diagnosis of rheumatic disorders involves distinguishing inflammation, degenerative changes and central sensitization from each other. Making this distinction is usually obvious or easily discernible on the basis of taking a history, performing a physical examination, viewing imaging studies as appropriate and obtaining laboratory analyses. There are occasions where traditional methods fail to either elicit a diagnosis or give the clinician clarity with regards to whether a patient, for example, is inflamed or has fibromyalgia (FM) .
Fibromyalgia has been a medical condition that for decades was relegated to being defined as a collection of subjective symptoms of varying characteristics lacking objective and reproducible laboratory features . FM is estimated to afflict a population in the USA alone of between 2 and 5 % of the US population . The annual cost of diagnosing FM makes it one of the most expensive diagnostic processes linked to any illness  as it presently chiefly relies on using tests to “rule out” disorders of rheumatic, neurologic, psychiatric, hematologic and endocrine origins. Hence, finding an objective diagnostic methodology offers substantial medical cost benefits. It additionally could lead to a potential way to monitor treatment efficacy as well as eliminate what has been an inexorable and prolonged pathway to an endpoint diagnosis that has previously utilized an indirect process of elimination.
In the last few years, laboratory panels have demonstrated that the degree or intensity of inflammation, for instance, may be further elucidated by composite scores or weighted testing that are statistically significant (e.g., multibiomarker disease activity <Vectra> for RA, cell-bound complement activation products <Avise> for SLE) [5, 6]. The multibiomarker disease activity index used in RA, for example, includes cytokine levels in its weighted scoring. In 2012, a group at the University of Illinois College of Medicine evaluated 8 cytokines and chemokines in 110 FM patients and 91 matched healthy controls . Unlike most previous studies, rather than measuring serum levels, peripheral blood mononuclear cells (PBMC) were stimulated by mitogens and in vitro production was quantitated. Statistically, significant findings distinguished the two groups as the FM group had 1.4-8 fold decreases in cytokine/chemokine production. Post hoc analyses determined that the panel was just as accurate if four chemokines and cytokines (instead of eight) were studied. Since FM is common in patients with autoimmune disorders, such as systemic lupus erythematosus (SLE) [8, 9], Sjogren’s Syndrome [10, 11] and rheumatoid arthritis (RA) , the chemokine/cytokine patterns found in FM patients may not have been unique to FM and were perhaps merely linked to rheumatic disorders as a whole, especially because of the frequent coexistence of FM and RA and FM and SLE. We therefore undertook comparative analyses to test the hypothesis that inflammation (represented by RA and SLE) and central sensitization syndromes (represented by FM) have distinct cytokine/chemokine signatures that could be clinically relevant.
Characteristics of patients
Fluorescence intensities were transferred into R (http:www.r-project.org) statistical software  for converting into concentration values. The standard curve was fitted with a 5-PL model, and the concentrations of the cytokines were quantified according to the curve. A two-sided t test with unequal variance was used to test whether the mean concentrations of each cytokine were the same in the groups. The descriptive statistics of the groups as well as the p values of the t test were calculated by using the stats package for R.
We used function lrm* from package rms  in R software to determine a logistical regression model on the concentration of the cytokines described above as predictor variables, and binary dependent variables and test scores were calculated.
Cytokine/chemokine composite (CCC) test scores of patients and healthy controls
95 % Confidence interval
CCC test score
−3.56 ± 5.7
−4.59 to −2.54
13 (11 %)
106 (89 %)
1.7 ± 1.2
1.52 to 1.89
149 (93 %)
11 (7 %)
−0.68 ± 2.26
−1.12 to −0.23
30 (31 %)
68 (69 %)
1.45 ± 3.34
−2.1 to −0.79
29 (29 %)
71 (71 %)
Characteristics of the CCC test scores in FM versus controls
Positive predictive value
Negative predictive value
Area under ROC curve
Wilcoxon test for FM scores versus controls
p < 2.2 × 10−16
Wilcoxon rank sum test of differences in stimulated cytokine/chemokine levels in SLE and RA samples compared to either FM samples or controls
Nearly 500 FM, autoimmune and healthy control patients underwent testing for cytokine/chemokine activity after mitogenic stimulation. Using a numerical score, all three groups had unique patterns with FM patients demonstrating less response to stimulation. This cytokine profile test had a 93 % sensitivity and an 89.4 % specificity for the diagnosis of FM. We also found that these profiles are relatively sensitive and specific for FM compared to SLE and RA. It remains unclear if these differences are directly related to the pathogenesis of FM.
We have been studying the role of cytokines in fibromyalgia for 30 years [17, 18]. Tumor necrosis factor and interleukins 1, 2, 6, 8 and 10 are associated with pain modulation, sleep induction, cognitive dysfunction, antinociception and sympathetic nervous system homeostasis to varying degrees. Studies assessing serum levels in our opinion are unreliable due to short half-lives, circadian rhythms (time of day that the measurements are obtained) as well as target tissue variance. Published reports have not demonstrated a consistent FM pattern. A few studies have looked at cytokine function after mitogenic stimulation. Patients with self-reported FM of <2 years duration had a greater response to stimulation than those with chronic FM . This is consistent with findings from the National Institutes of Health that although serum cortisol levels are normal in the syndrome, response to cortrosyn stimulation is blunted . Further work has suggested that sympathetic nervous system responses are less robust in FM when ascertained by decreased heart rate variability or exercise [19, 20].
The scores found in this report are consistent with the finding that chronic stress decreases cytokine/chemokine responses. IL-6 is an acute phase reactant that is associated with stress, fatigue, hyperalgesia and sympathetic nervous system activation; IL-8 induces chemotaxis, phagocytosis, angiogenesis and modulates sympathetic mediated pain, and MIP-1 alpha and beta recruit polymorphonuclear cells and are chemoattractants for natural killer cells and monocytes. In all probability, chronic sensitization syndromes (which includes FM) are associated with a milieu whereby responses to sympathetic, hormonal, cytokine and chemokine stimulation are diminished. In the last decade, it has also become evident that glial cells produce cytokines, and complex interactions in the setting of FM might explain opioid induced hyperalgesia observed in the syndrome . In other words, FM patients often fare worse when prescribed narcotic analgesics.
The expression of serum cytokines and chemokines in RA and SLE is increased, and the use of anticytokine therapies (and Phase 1 and 2 studies of chemokine inhibition) for autoimmune disease suggests that the findings would be opposite of what has been shown in FM .
Our study was confounded by different times of day that the cytokine/chemokine profile was obtained, concomitant anti-inflammatory and immune modulating medications, the use of corticosteroids and coexistence of FM in <10 % of the autoimmune (SLE and RA) patients. Despite this, we were able to demonstrate statistically significant differences in scores comparing patients with FM, healthy controls and autoimmune disease. The study was exploratory and provided preliminary information documenting a signal. Now that there is one, further data mining and/or a more focused second study would allow us to examine which subsets or autoimmune “phenotypes” are more likely to influence a combined chemokine score. This would, for example, include looking at established disease activity indices and acute phase reactants (e.g., sedimentation rate, C reactive protein). High cytokine/chemokine composite test scores strongly suggest a central sensitization component with or without an inflammatory condition. We posit that using a cytokine/chemokine stimulated response composite score is clinically useful in the differential diagnosis of FM patients as well as in patients where the role of inflammation versus central sensitization would benefit from further delineation. For example, changes in the score could be used to monitor response to different interventions.
Dr. Gillis personally underwrote the study and owns EpicGenetics. Dr. Wallace was a consultant for EpicGenetics in 2013. Dr. Gavin, Karpenko and Barkhordar are employees of EpicGenetics.
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