Background

Fibromyalgia syndrome (FMS) is a complex of symptoms that, according to the criteria of the American College of Rheumatology (ACR), is associated with chronic widespread pain and at least eleven positive tender points out of 18 [1]. Additional symptoms like fatigue, pain associated sleep disturbance, depression, or gastrointestinal disorders are frequently reported [2]. The pathophysiology of FMS is incompletely understood and although the syndrome can be characterized by an identifiable group of signs and symptoms, there is no recognized etiologic agent and no consistent anatomical alteration that would qualify FMS as a disease (e.g. Stedman's online medical dictionary http://www.stedmans.com).

With an estimated prevalence of 0.5% to 5.8% in the general population of North America and Europe [3, 4], FMS is a frequent condition and better pathophysiological knowledge is warranted to more effectively direct treatment. Although a large number of studies on the pathophysiology of FMS have been published, there is still no overall concept explaining all aspects of FMS. An imbalance of pro- and anti-inflammatory cytokines is assumed to play a role in the induction and maintenance of pain [5] and several laboratories have investigated cytokine levels in patients with FMS with diverse results. In non-systematic reviews the potential pathophysiological role of cytokines has been discussed with discrepant results [6, 7]. A systematic review with a quantitative synthesis of the cytokine profiles of FMS patients compared to controls had not been performed until now. The aims of this systematic review therefore were to systematically and quantitatively review data on pro- and anti-inflammatory cytokines in FMS patients considering and assessing study quality.

Methods

Setting

The literature search was initiated as part of the development of an evidence-based interdisciplinary guideline for the diagnosis and therapy of FMS on behalf of the Association of the Scientific Medical Societies of Germany (AWMF) coordinated by the German Interdisciplinary Association of Pain (DIVS) [8]. The search was later expanded and extended (see below).

Searches

A literature search for original research articles on the pathophysiology of FMS was performed through December 2010 and the following databases were screened: PubMed, MEDLINE, and PsycINFO. The keywords (all languages) "fibromyalgia", "fibromyalgia syndrome", "chronic widespread pain" were used in combination with "cytokine", with "cytokine" AND "pathophysiology", and also with "review". Additionally, the reference sections of 22 review articles that were found using this search strategy were screened for possibly suitable original articles.

Inclusion and exclusion criteria

We only included original full text articles investigating the pathophysiological role of cytokines in FMS and excluded the following articles that appeared upon the search strategy detailed above: double hits, reviews, letters to the editor, papers investigating other disorders, articles in Danish, all articles that did not investigate cytokines. First, the titles and abstracts of all articles found were screened for suitability; the initially chosen articles were then screened again checking the entire article.

Data extraction

Two authors independently screened the titles and abstracts of potentially eligible articles that were detected by the search strategy described above. The full text of the selected studies was examined. For data extraction two authors (NÜ, CS) independently used standard extraction forms. All authors cross-checked the extraction forms for correctness. Data were categorized for the material investigated. Blood measurements were performed in serum, plasma, and whole blood; accordingly these data were first assessed separately and then all study results on "blood" were compared.

Quality assessment

For evaluating the quality of the selected studies we applied two validated scales: the Level of Evidence of the Oxford Center for Evidence-based Medicine (CEBM, http://www.cebm.net/index.aspx?o=1025) and the Newcastle-Ottawa Quality Assessment Scale (NOS) http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp.

We modified the CEBM to make it applicable for scientific papers investigating pathophysiological aspects with bench research (Table 1). Additionally, we aimed at evaluating the quality of the laboratory methods used. To our knowledge there is no standardized rating scale to assess the quality of laboratory methods in scientific papers. Therefore we created the Würzburg Methodological Quality Score (W-MeQS, Table 2). W-MeQS consists of twelve items that are relevant for high quality of a laboratory method. For each item fulfilled one point is given; the score is the sum of all points achieved divided by the number of items that were applicable. If the target of interest was measured by just one method this score marks the end score. If more than one method was used, a subscore is calculated for each method as described and the end-score is calculated as mean of the subscores. The scores were evaluated as follows: ≤ 0.4: low quality; > 0.4 medium quality; ≥ 0.8 high quality.

Table 1 Levels of CEBM modified for scientific papers investigating disease pathophysiology with bench research.
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Table 2 Würzburg Methodological Quality Score (W-MeQS) for the assessment of the quality of laboratory methods.
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Data synthesis and analysis

Meta-analysis was performed using RevMan analysis software (RevMan 5.1.2) of the Cochrane Collaboration [9]. Standardized mean differences (SMD) were calculated by means and standard deviations for each cytokine.

Results

Study selection

For the number of found, selected, excluded and finally included articles see the algorithm shown in Figure 1.

Figure 1
figure 1

Algorithm of study selection. The Figure displays the details of the study search and selection process.

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The initial literature search retrieved the following numbers of articles: PubMed: 281; MEDLINE: 69; PsycINFO: 2. After exclusion of double hits 115 articles were chosen and the titles and abstracts were screened for suitability. The following 90 articles were excluded: reviews (n = 42), papers investigating other disorders (n = 26), papers not investigating cytokines (n = 11), letters to the editor (n = 4), double publications (n = 3), treatment studies (n = 3), and one article in Danish. The screening of the reference sections of 22 review articles did not provide further original articles. Finally, 25 articles were selected for our systematic review[10–34]. A total of 1255 patients with FMS, 180 patients in disease control groups, and 800 healthy controls were investigated.

Of these 25 articles, two articles were not suitable for meta-analysis because no healthy control group was investigated [11, 26]; seven articles could not be included, because mean and standard deviations were not given in the article or could not be calculated (e.g. from the standard error of the mean) and were not provided upon request [10, 13, 15, 20, 21, 25, 27]. Finally, 16 articles were principally suitable for meta-analysis[12, 14, 16–19, 22–24, 28–34]. These articles were then screened for suitability to be categorized in groups of articles having investigated the same cytokine with the same method in the same material; three of the 16 articles could not be categorized in one of the subgroups (see below) and therefore could not enter meta-analysis [12, 18, 33]. Thus, 13 articles were finally included in the meta-analysis. Table 3 summarizes key information of the 25 selected articles and gives the quality scores of the studies.

Table 3 Characteristics of all studies included in the systematic review investigating cytokines in FMS patients.
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Majority of studies with low quality

Only three of the 25 studies (12%) had a modified CEBM level of 3c [14, 15, 19]. Twenty-one of the 25 (84%) studies had a modified CEBM level of 3d [10–13, 16–18, 20–25, 27–34], and one case report of 4 (4%) [26]. The highest NOS score reached was 4 out of 8; this was achieved by 5/25 studies (25%) [12, 28, 30, 32]. Eleven out of 25 studies (44%) reached a NOS score of 3 [11, 13–17, 20, 24, 25, 31, 33]; 8/25 studies (32%) had a NOS score of 2 [10, 18, 21–23, 27, 29, 34]; one study had a NOS score of 1 (4%) [26].

In the W-MeQS only 4/25 studies (16%) were of high methodological quality (score ≥ 0.8) [14, 15, 29, 30]; 7/25 studies (28%) were of medium quality (score > 0.4) [10, 19–21, 27, 31, 33], and 14/25 studies (56%) were of low quality (score ≤ 0.4) [11–13, 16–18, 22–26, 28, 32]. Only 2/25 studies (8%) had a high NOS and W-MeQS score [14, 15].

These results show that the majority of the studies were of low methodological quality.

High variability of materials and methods

In twelve studies serum [11, 13, 19, 20, 23, 25, 26, 28, 30–32, 34], in seven studies peripheral blood mononuclear cells (PBMC) [10, 12, 18, 21, 22, 24, 31], in six studies plasma [13, 14, 16, 17, 29, 33], in two studies skin biopsies [15, 27], and in one study whole blood [30] was investigated. Cytokine levels were investigated with ELISA in twelve studies [11–14, 19, 23–25, 28, 30, 31, 34], with bead-based immunoassays in five studies [17, 20, 26, 29, 33], with FACS analysis in two studies [10, 21], with quantitative real-time PCR in one study [30], with reverse transcription PCR in one study [27], with immunohistochemistry in one study [15].

There is little overlap in materials and methods used between different studies investigating cytokine levels in FMS patients.

Systematic review

Serum (Table 4)

Serum samples of 643 FMS patients, 109 patients in disease control groups, and 301 healthy controls were investigated. Only one study reached a modified CEMB level of 3c [19]; ten studies had a modified CEMB level of 3d [11, 13, 20, 23, 25, 28, 30–32, 34]; one study had a modified CEMB level of 4 [26]. Four studies had a NOS score of 4 [19, 28, 30, 32], five studies had a NOS score of 3 [11, 13, 20, 25, 31], two studies had a NOS score of 2 [23, 34], and one study of 1 [26]. Applying W-MeQS only one study was of high methodological quality [30]; three studies were of medium quality [19, 20, 31], and eight studies of low methodological quality [11, 13, 23, 25, 26, 28, 32, 34].

Table 4 Characteristics of studies investigating serum levels of cytokines in FMS patients.
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The following results were reproduced (regardless of the method used and with a minimum of two independent studies): patients with FMS have higher serum levels of IL-1RA [20, 23, 31], IL-6 [19, 20, 28], and IL-8 [32, 34]. No intergroup difference was found for serum IL-6 [13, 23, 25, 31, 32, 34], IL-1β/IL-1 [13, 20, 25, 31, 34], TNF [13, 25, 30–32], IL-2 [30, 31], IL-10 [13, 31, 32], IFN-γ [20, 31], IL-4 [20, 32].

Discrepant results are described for IL-6: IL-6 levels were found higher in FMS patients compared to controls in three studies [19, 20, 28]; 2/3 studies were of medium methodological quality [19, 20], 2/3 studies had a NOS score of 4 [19, 28], and 1/3 study had a modified CEBM of 3c [19]. In six studies no intergroup difference could be found for serum IL-6 levels [13, 23, 25, 31, 32, 34]; 5/6 studies were of low methodological quality [13, 23, 25, 32, 34], only 1/6 studies reached a NOS score of 4 [32], and 6/6 studies had a modified CEBM level of 3d [13, 23, 25, 31, 32, 34]. The better quality of studies showing elevated serum IL-6 levels in FMS patients compared to controls favors this finding, although in more studies (with less quality) no intergroup difference could be found.

The majority of studies were of low methodological quality and there was hardly an overlap in the methods used and the cytokines investigated. Only IL-1RA, IL-6, and IL-8 were higher expressed in sera of FMS patients, while for all other cytokines no intergroup difference could be found.

Plasma (Table 5)

Plasma cytokine levels were investigated in seven studies [13, 14, 16, 17, 29, 31, 33].

Table 5 Characteristics of studies investigating plasma levels of cytokines in FMS patients.
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Plasma samples of 699 FMS patients, 109 patients in disease control groups, and 357 healthy controls were investigated. Only one study reached a modified CEMB level of 3c [14]; six studies had a modified CEMB level of 3d [13, 16, 17, 29, 31, 33]. Six studies had a NOS score of 3 [13, 14, 16, 17, 31, 33], one study of 2 [29]. Applying W-MeQS only two studies were of high methodological quality [14, 29], two studies were of medium [31, 33], and two studies of low methodological quality [16, 17].

The following results were reproduced (regardless of the method used and with a minimum of two independent studies): patients with FMS have higher plasma levels of IL-8 [14, 31]. No intergroup difference was found for plasma IL-1β/IL-1 [13, 14, 17, 29, 31, 33], IL-6 [13, 14, 29, 31, 33], IL-8 [13, 16, 29, 33], TNF [13, 16, 29, 31, 33], and IL-10 [13, 29, 31, 33], MCP-1 and IP10 [17, 33], IL-2 and IFN-γ [31, 33].

Discrepant results are described for IL-8: IL-8 levels were found higher in FMS patients compared to controls in two studies [14, 31]; 1/2 studies was of medium [31] and 1/2 studies was of high methodological quality [14], both studies had a NOS score of 3, and one study reached a modified CEBM level of 3c [14]. In four studies no intergroup difference could be found for plasma IL-8 levels [13, 16, 29, 33]; 2/4 studies were of low methodological quality [13, 16] and 3/4 studies had a NOS score of 3 [13, 16, 33]; the only study with high methodological quality had a NOS score of only 2 [29]; all four studies had a modified CEBM level of 3d. The better quality of studies showing elevated plasma IL-8 levels in FMS patients compared to controls favors this finding, although in more studies (with less quality) no intergroup difference could be found.

The majority of studies were of low methodological quality with only little overlap in methodology and cytokines investigated. A difference between FMS patients and controls was found only for IL-8 plasma levels. When taking studies investigating serum and plasma together we found no intergroup difference.

Whole blood (Table 6)

Cytokine expression in whole blood of patients with FMS was investigated in one study including 26 FMS patients and 40 healthy controls [30]. The study had a modified CEMB level of 3d, a NOS of 4 and was of high methodological quality and found reduced levels of the anti-inflammatory cytokines IL-4 and IL-10 gene expression in FMS patients compared to controls. These results were not reproduced.

Table 6 Characteristics of studies investigating whole blood levels of cytokines in FMS patients.
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Peripheral blood mononuclear cells (Table 7)

Cytokine expression in peripheral blood mononuclear cells (PBMC) was investigated in seven studies [10, 12, 18, 21, 22, 24, 31].

Table 7 Characteristics of studies investigating levels of cytokines in PBMC of FMS patients.
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PBMC of 151 FMS patients, 21 patients in disease control groups, and 159 healthy controls were investigated. All seven studies had a modified CEMB level of 3d [10, 12, 18, 21, 22, 24, 31]. One study reached a NOS score of 4 [12], two studies of 3 [24, 31], and four studies of 2 [10, 18, 21, 22]. Applying W-MeQS three studies were of medium methodological quality [10, 21, 31] and four were of low quality [12, 18, 22, 24].

The following results were reproduced (regardless of the method used and with a minimum of two independent studies): no intergroup difference was found for IL-10 secretion from PBMC between FMS patients and controls [10, 22, 31].

FMS patients do not differ in PBMC cytokine levels from healthy controls.

Skin (Table 8)

Cytokine expression in skin of FMS patients was investigated in two studies including 116 FMS patients, and 59 healthy controls [15, 27]. One study had a modified CEMB level of 3c [15] and one of 3d [27]; one study had a NOS score of 3 [16], the other of 2 [27]. Applying W-MeQS one study was of high methodological quality [16], one study of medium quality [27]. In the study by Salemi et al. [27] IL-1β, IL-6, and TNF were detectable in skin samples of a subgroup of FMS patients using reverse transcription PCR and immunohistochemistry, while in controls none of these cytokines could be detected. Blanco et al. [15] investigated TNF and MCP-1 in skin biopsies using immunohistochemistry and found lower MCP-1 expression in FMS patients than in controls. None of the results were reproduced.

Table 8 Characteristics of studies investigating levels of cytokines in skin of FMS patients.
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Meta-analysis

In the selected 16 papers for meta-analysis authors investigated different cytokines with various methods. We therefore created subgroups of papers that investigated the same cytokine with the same or a comparable method. A group minimum of three studies was mandatory for meta-analysis.

A significant difference between controls and FMS was found only in two subgroups. When analyzing data of three studies on the pro-inflammatory cytokine IL-6 plasma levels investigated with ELISA or immunoassay or bioplex assay, patients with FMS had higher plasma levels of IL-6 compared to controls (SMD, -0.34; [-0.64, -0.03] 95% CI; p = 0.03; Table 4). There was also a trend for elevated serum IL-6 levels in FMS patients compared to controls (SMD, 1.01; [-0.03, 2.05] 95% [CI]; p = 0.06). No further intergroup differences were found even when regarding studies investigating serum, plasma, and whole blood as one group of "blood" (Table 9).

Table 9 Results of meta-analysis.
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Patients with FMS have higher plasma levels of IL-6 compared to controls. The discrepancy to the results of the systematic review (higher plasma IL-8 levels in patients with FMS) is due to the fact that only few studies were suitable for meta-analysis.

Discussion

In this systematic review and meta-analysis we set out to clarify if cytokine profiles in FMS patients differ from controls analyzing published data available to date. Applying the modified CEBM levels, NOS, and W-MeQS we showed that the majority of the studies was of poor methodological quality. Our systematic review, in which we also considered study quality (modified CEBM level; NOS score; W-MeQS) shows that patients with FMS have higher serum IL-1RA, IL-6, and IL-8 levels compared to controls and that they also have higher plasma levels of IL-8. Looking at reproduced results (regardless of the method used and with a minimum of two independent studies) FMS patients do not differ from controls in serum IL-1β/IL-1, TNF, IL-2, IL-10, IFN-γ, and IL-4 levels; they also do not differ from controls in plasma IL-1β/IL-1, IL-6, TNF, IL-10, MCP-1, IP10, IL-2, and IFN-γ levels.

Our meta-analysis shows that patients with FMS have higher plasma levels of IL-6 compared to controls and a tendency for higher serum levels of IL-6 (p = 0.06). No further differences could be found.

IL-1RA, IL-6, and IL-8 are pro-inflammatory cytokines that may have algesic effects. The role of cytokines in the induction and maintenance of pain is well established in animal studies and also in pain syndromes [5]. Therefore the hypothesis that patients with FMS, who suffer from generalized pain may have an innate or acquired imbalance in cytokine production and secretion is plausible. Higher levels of these cytokines in plasma and/or serum of patients with FMS may be associated with pain in FMS. In line with these findings one study showed lower levels of the anti-inflammatory and analgesic cytokines IL-4 and IL-10 in FMS patients compared to healthy controls [30]. However, it remains elusive, whether these systemic changes in cytokine levels are the cause of pain in FMS or its consequence. Longitudinal studies are needed to answer this question.

The main problem in interpreting the results of studies on cytokines in FMS is that although cytokines have been investigated in a large number of studies, different methods have been used to analyze different cytokines in diverse material. Therefore a direct comparison of the results is hardly possible. Results obtained for one cytokine have mostly not been reproduced, the number of samples investigated per study was low (7/25 studies with n < 20 patients), and several studies report conflicting results - even in the same research group [13, 14]. Also, possible confounding factors that physiologically may influence cytokine levels (e.g. circadian production; depression; physical activity; current infection) were not controlled in the majority of the studies.

Another problem is that the measurements mostly were not specifically hypothesis-driven, but were performed following the general assumption that "cytokines may play a role". Therefore in the majority of studies a large array of cytokines was screened without a clear hypothesis for each cytokine. This might be one reason why only few markers were repetitively analyzed, however, most markers were investigated only once - probably because they were part of a commercially available kit. In none of the studies a mechanistical approach was followed to investigate the mechanisms possibly underlying the effect of a specific cytokine in the induction and maintenance of defined symptoms of FMS. Therefore also the biological impact of the described changes in cytokine levels remains elusive. The question is also if in all studies the most appropriate method was used to detect the cytokine of interest. The majority of cytokines have a low expression and it is essential to apply methods that are sensitive enough. In the last years high- and ultra-sensitive bioassays have been developed that reliably allow the determination of cytokine proteins in body fluids. Since in the majority of cytokine studies analyzed here the sensitivity of the used method was not given it is not clear if the many negative results reported are "real" or due to low method sensitivity.

One further point is that there is no validated scoring system for the quality of laboratory methods in research papers. Such tools, however, are needed to assess study results and to compare the results of different studies, especially when looking at conflicting results. Standardization of the way laboratory research is performed by following quality standards and its presentation is warranted. The quality score W-MeQS that we present here may be one possibility.

In many cases the data provided in the papers were not applicable for meta-analysis and could not be calculated from the data presented in the publication. This fact and the diversity of methods and materials investigated made it almost impossible to form subgroups for meta-analysis.

Cytokines form a "fragile" system that can be influenced by many factors. The following issues might help improving the quality and the yield of future cytokine research in FMS patients: 1) Cytokine secretion follows circadian rhythmicity; the time points of material asservation should be kept constant. 2) Cytokine secretion has a large variability; measurements should be performed on two sets of samples and at least two different methods should be used (e.g. investigation of gene expression and protein levels). 3) Cytokine levels can be influenced by many environmental factors and by medication; patients should therefore be off medication at the time of material asservation or the possible influence of medication should be considered when interpreting the results. 4) Detailed patient characteristics (including disease duration; pattern of symptoms etc.) are essential for correlation of the cytokine measurements with clinical data. 5) The formulation of a clear literature-based hypothesis is warranted at the beginning of the cytokine study and a power calculation should be performed in advance. 6) Systemic and local cytokine expression should be investigated with appropriate methods. To choose the appropriate method, the expected level of expression should be taken into account and it should be made sure that the sensitivity of the method used is high enough to measure what is to be measured.

Conclusions

The major consequence of our review is that cytokine research in FMS pathophysiology needs substantial improvement. More hypothesis-based and mechanistic studies are needed to understand if distinct cytokines are involved in causing symptoms of FMS or if they may be used as biomarkers of FMS symptoms. There is also a need for defined and applied quality standards for study design and performance in bench-side research and for standardization rules for study reports and manuscripts (like STROBE for clinical research). Possible confounding factors of cytokine levels should be controlled and a sufficient number of study subjects should be investigated e.g. following an a priori power calculation of sample size. Also the performance of prospective studies would be helpful.