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. 2008 Dec;154(3):346–352. doi: 10.1111/j.1365-2249.2008.03787.x

May genetic factors in fibromyalgia help to identify patients with differentially altered frequencies of immune cells?

L S C Carvalho *, H Correa †, G C Silva †, F S Campos *, F R Baião ‡, L S Ribeiro §, A M Faria , D d'Avila Reis *
PMCID: PMC2633236  PMID: 19037919

Abstract

There is common agreement that fibromyalgia (FM) is an extremely heterogeneous entity. Patients differ in their clinical symptoms, endocrine and immune parameters. In this study we evaluated endocrine and immunological features of distinct subsets of FM patients. In contrast to previous attempts to identify subsets of FM patients, based solely on their psychological and cognitive features, herein we propose to separate FM patients by genetic features. Allelic expression of the polymorphic promoter region of the serotonin transporter (5-HTTLPR) was analysed as a relevant genetic factor for FM. Seventy-five patients meeting the American College of Rheumatology criteria and 27 healthy age-matched controls participated in this study. All controls and FM patients were submitted to genotyping of 5-HTTLPR. Twenty-seven FM patients, who were able to discontinue hypnotic, sedative or psychotropic prescription medications for at least 2 weeks, were then subdivided into L (homozygote LL) or S groups (genotypes LS and SS). They were evaluated for salivary cortisol levels, absolute number of leucocyte subpopulations, including natural killer (NK) cells and activated T and B lymphocytes. Both groups presented decreased cortisol levels, more intense in the L group, increased all B lymphocytes subsets and reduced CD4+CD25high T lymphocytes. The L group had increased CD4+CD25low activated T lymphocytes, while the S group displayed elevated CD4+human leucocyte antigen D-related (HLA-DR)+ activated T lymphocytes and decreased NK cells. We demonstrate that genetic factors may help to identify FM individuals with differentially altered frequencies of immune cells.

Keywords: fibromyalgia, genetic, 5-HTTLPR, lymphocytes, salivary cortisol

Introduction

Fibromyalgia (FM) is a chronic syndrome characterized by widespread joint and muscle pain accompanied frequently by other clinical features, such as depression, anxiety and sleep disturbances [1]. These characteristics, however, typically exhibit considerable variation, and clinical presentation, course and prognosis can be very different between patients, suggesting that FM is a heterogeneous syndrome. Following the American College of Rheumatology (ACR) diagnosis, criteria include diffuse soft tissue pain lasting at least 3 months and pain on palpation in at least 11 of 18 paired tender points [1].

The aetiology of FM is still unclear and the literature is somewhat contradictory; nevertheless, it appears that the immune system could be involved either directly or as a result of a dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis and/or of the sympathetic nervous system (SNS) [2–6]. First, some authors have described increased levels of proinflammatory cytokines [3] and activated T lymphocytes in FM patients [4], while others have failed to demonstrate any immunological change associated with this chronic syndrome [7,8]. Additionally, the presence of autoantibodies in the blood of FM patients has been described, as well as the association of this syndrome with other already described autoimmune diseases, such as rheumatoid arthritis, Hashimoto's thyroiditis and enthesopathies [5,6,9,10]. Secondly, mild hypocortisolaemia has been observed by some investigators [11–15], while others report normal patterns or increased cortisol levels in FM patients [16–18].

A remarkable feature of this syndrome is the great variability in the clinical, immunological and endocrine data collected from FM patients. Our working hypothesis is that such variability could be related, at least in part, to the existence of distinct FM groups according to genetic markers. In fact, genetic factors may play a role in the aetiopathology of FM, as there is a high aggregation of FM in families, and there is evidence that polymorphisms of genes in the serotoninergic, dopaminergic and catecholaminergic systems are associated with FM [19–22].

The serotonin transporter gene is of particular interest because the magnitude and duration of serotoninergic activity is regulated mainly by the human serotonin transporter (5-HTT), which controls the uptake of serotonin from the synaptic junction [23,24]. There is some evidence showing that serotonin transporter may be involved in FM. A recent study showed that changes in the platelet serotonin transporter appear to occur in fibromyalgic patients, apparently related to the severity of fibromyalgic symptoms [25]. Furthermore, in the promoter region of serotonin transporter there are two allelic variants, a long (L) variant and a short (S) variant that differ in length by 44 base pairs (bp), named the 5-HTTLPR polymorphism. In vitro studies show that the L allele has two to three times higher basal transcriptional activity. The homozygous LL genotype polymorphism has been shown to have higher transcriptional activity for the 5-HTTLPR in vitro than genotypes containing one or two S alleles [26]. Some authors demonstrated a higher frequency of the S/S genotype in FM patients compared with healthy controls, data that were confirmed later by Cohen et al.[27,28]. They showed further that the S/S group exhibited higher mean levels of depression and psychological distress [27]. Taken together, these results suggest that serotoninergic dysfunction may play a role in this syndrome, and they also point to the possible existence of two distinct genetic subpopulations of FM patients.

Because several reports have described discrepant immunological changes in FM our hypothesis was that such heterogeneity could be explained, at least in part, by the existence of distinct genetic populations. In order to investigate this hypothesis, we grouped FM patients according to the 5-HTTLPR genotype, and investigated if the two groups presented different immune disturbances when compared with control individuals. We evaluated the absolute number of leucocyte subpopulations, including natural killer (NK) cells, T and B lymphocytes, CD4+CD25high T lymphocytes and activated T and B lymphocytes.

Materials and methods

Patients and controls

Seventy-five patients with FM participated in this study. They were diagnosed according to ACR criteria by a specialized clinician at Baleia Hospital and Instituto de Previdência dos Servidores do Estado de Minas Gerais, Belo Horizonte, MG, Brazil. All patients were submitted to 5-HTTLPR genotyping. Twenty-seven patients who were able to discontinue hypnotic, sedative or psychotropic prescription medications for at least 2 weeks were also analysed for salivary cortisol and immunological parameters, as described below. For comparative analysis this group of 27 patients was split according to 5-HTTLPR genotyping as the L group (patients who expressed only the long allele) and the S group (patients with at least one short allele). The use of birth control pills, menopausal and dipyrone or acetaminophen for pain relief was permitted. Twenty-seven age- and sex-matched healthy adults were selected for the control group. They had negative past, present or family history for psychiatric disorders.

The study was approved by the Human Research Committee of Federal University of Minas Gerais and Governador Israel Pinheiro Hospital. All patients and control subjects gave written informed consent before inclusion in the study.

Blood samples and white blood cell counts

A 5-ml sample of peripheral blood was collected from each subject using ethylenediamine tetraacetic acid as the anti-coagulant. After collection, leucograms were performed by haemocytometer counts (Coulter®Ac·TdiffTM; Beckman Coulter, Fullerton, CA, USA) and the numbers of lymphocytes and monocytes were calculated by counting cells on air-dried films stained with May–Grunwald–Giemsa solutions (Doles Reagentes, Goiania, GO, Brazil).

Flow cytometry

Mouse anti-human monoclonal antibodies (mAbs) conjugated with phycoerythrin (PE), fluorescein isothiocyanate (FITC) or Cychrome (Cy) were used for two- or three-colour flow cytometric assay. In this study, we used anti-human FITC conjugated mAbs including anti-CD3, anti-CD4, anti-CD8, anti-CD5, anti-CD19 and anti-CD16. The following second-colour reagents were used: anti-human PE-conjugated mAbs anti-CD3, anti-CD4, anti-CD8, anti-human leucocyte antigen D-related (HLA-DR), anti-CD25, anti-CD69, anti-CD19 and anti-CD23. As third-colour reagent, anti-human Cy-conjugated mAbs anti-CD56 were used. Non-related immunoglobulin (Ig)G mAbs (IgG1-FITC and IgG1-PE) were used at the same concentrations as non-specific binding controls. All mAbs used in this study were purchased from Pharmingen (San Diego, CA, USA).

The antibodies were diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin. Immunofluorescence assays were performed by incubating 50 µl of whole blood with 10 µl of diluted mAbs in the dark for 30 min at room temperature. After incubation, erythrocytes were lysed with lysing solution (Becton Dickinson, San Jose, CA, USA) and washed with PBS. Cells were fixed with 200 µl of 1% paraformaldehyde, 1% sodium cacodylate/NaCl. Flow cytometry data acquisition was performed with a Becton Dickinson fluorescence activated cell sorter (FACScan) instrument. A total of 10 000 events per tube were acquired and analysed using CellQuest software. Results were expressed in absolute numbers on the basis of the percentage of expression of the different markers and the total numbers of lymphocytes on peripheral blood.

Measuring of cortisol

Salivary cortisol level was used as an objective marker of HPA axis activity. Three saliva samples from patients and the control group were collected using cotton rolls at 8 a.m., 5 p.m. and 10 p.m. 1 day before blood samples were collected. Subjects were instructed to not take food or brush their teeth 30 min before sample collection to avoid contamination of saliva samples, to store saliva samples in their freezers until completing the experimental protocol, and then return the samples to the laboratory. Samples were centrifuged and frozen at −20°C. Free cortisol in saliva was determined using a time-resolved immunoassay according to the manufacturer's instructions (DSL-10 67100 Diagnostic Systems Laboratories, Webster, TX, USA). The sensitivity of this test was 0·30 nmol/l. The intra- and interassay coefficients of variation were 3·5% and 5·1% respectively.

Genotyping

Genotyping was performed as described previously [29], with slight modifications. Briefly, after genomic DNA extraction from a 5 ml peripheral blood sample the 5-HTT regulatory gene region was amplified using polymerase chain reaction (PCR) with the oligonucleotide sense primer 5′-CCGCTCTGAATGCCAGCACCTAAC-3′ and anti-sense primer 5′-AGAGGGACTGAGCTGGACAACCAC-3′. The PCR product yielded 478/522 bp fragments that were resolved by electrophoresis in 8% polyacrylamide gel and silver-stained to ascertain the exact size of the amplified products.

Mood state

A major depression episode diagnosis was conduced by a psychiatrist using a structured interview (MINI-PLUS) following DSM-IV criteria, blinded to genetic/immunological results. The Portuguese version of Spielberger's state–trait anxiety inventory (STAI) [30] was used to assess anxiety in patients and control subjects. The X1 questionnaire served as an indicator of state anxiety; the X2 questionnaire was used to assess trait anxiety. Both questionnaires were used in the interview.

Statistics

Data were analysed by analysis of variance and Tukey's post hoc test using the Graphpad Prism software release 4·02 for Windows (San Diego, CA, USA). Categorical data were analysed using the χ2 test. Data were expressed as the mean ± standard error. Significance was defined as P ≤ 0·05.

Results

Clinical features

This study was conducted with the participation of 71 females and four males in the FM group, and 25 females and two males in the control group. There were no statistically significant differences in age or body mass index (BMI) (Table 1). All patients were negative for hepatitis C virus infection. The frequency of major depression (past and/or actual episode) was 29% and 65% of patients in the L and S groups respectively. These patients showed some depression mood state, more intense in the S group (χ2 = 5·11, d.f. = 1, P = 0·03). However, both fibromyalgic patient groups presented increased scores on the STAI scale trait (P = 0·009) and state of anxiety (P = 0·008) when compared with the control group (Table 1).

Table 1.

Clinical features and mood state in fibromyalgia (FM) patients and controls.

Controls (n = 27) FM patients
All (n = 75) L group (n = 25) S group (n = 50)
Age (years) 44 ± 2 50 ± 1 47 ± 2 51 ± 1
BMI (kg/m2) 25 ± 1 27 ± 1 25 ± 3 28 ± 1
Illness duration (years) – 4 ± 0·5 4 ± 1 4 ± 0·5
Depression – 50% 29%** 65%**
STAI: trait score 36 ± 2 47 ± 1* 46 ± 1* 46 ± 1*
STAI: state score 36 ± 4 46 ± 1* 46 ± 1* 46 ± 1*
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*

P < 0·05 between patients and controls;

**

P < 0·05 between patient groups. Variables were expressed as mean ± standard error. BMI, body mass index. L group, homozygote LL; S group, genotypes LS and SS; STAI, state–trait anxiety inventory.

Promoter region genotype of the serotonin transporter gene

Genotypic distribution was significantly different between healthy controls (LL = 11, 52%; LS = 8, 38%; SS = 2, 10%) versus FM patients (LL = 25, 33·3%; LS = 27, 36%; SS = 23, 30·7%) (χ2 = 14·29, d.f. = 2, P = 0·008) with clear over-expression of the SS genotype in FM patients. This difference could not be explained by the demographic parameters we studied, as no differences were found between healthy controls and patients concerning age, gender distribution and BMI. Nevertheless, FM patients presented increased scores on STAI scale trait and state of anxiety (Table 1). Healthy controls (χ2 = 0·09, P = 0·76) but not patients (χ2 = 5·86, P = 0·016) were in Hardy–Weinberg equilibrium.

When patients were separated further according to the functionality of the genotypes (LL versus LS+ SS genotypes), healthy controls and FM patients still presented statistically significant differences (χ2 = 178, d.f. = 1, P = 0·0001). This difference could not be explained by the demographic parameters we studied, because no differences were found between the two groups concerning age, gender distribution and BMI (see Table 1). Increased frequency of major depression (past and/or actual episode) was presented by the S group (65%) when compared with the L group (29%) (see Table 1).

Enumeration of peripheral blood leucocyte subpopulations

The FM patients in the L group presented marginally significantly increased numbers of neutrophils when compared with the healthy control group (4620 ± 413 cells/mm3 and 3710 ± 429 cells/mm3versus 3520 ± 260 cells/mm3 for the L, S and control groups respectively; P = 0·06 for the L group compared with the control group). No significant alterations were observed in the numbers of total lymphocytes, total eosinophils or total monocytes in the FM patients (data not shown).

Subpopulations of T lymphocytes, B lymphocytes and NK cells

Compared with healthy controls, no significant difference was observed in the number of either CD3+CD4+ or CD3+CD8+ T lymphocytes in FM patients (Fig. 1). The analysis of CD4+CD25high T cells, a phenotype usually associated in humans with regulatory T lymphocytes, revealed reduction of this cellular population in both groups of patients (8 ± 1 cells/mm3 and 5 ± 2 cells/mm3versus 76 ± 14 cells/mm3 for the L, S and control groups respectively, P = 0·002).

Fig. 1.

Fig. 1

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Absolute numbers of CD3+ T lymphocytes, CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes on peripheral blood of controls (grey bars), homozygote LL (L group) of fibromyalgia (FM) patients (hatched bars), and genotypes LS and SS (S group) of FM patients (black bars). The results were expressed as mean ± standard error.

Double-labelling studies using anti-CD19-PE and anti-CD5-FITC were used to determine the number of total B lymphocytes (CD19+), B1 subset (CD19+CD5+) and B2 subset (CD19+CD5−). The three cellular populations were found increased significantly in both the L and S groups, when compared with matched controls (Fig. 2).

Fig. 2.

Fig. 2

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Absolute numbers of CD19+ B lymphocytes, CD19+CD5+ B1 lymphocytes and CD19+CD5− B2 lymphocytes on peripheral blood of controls (grey bars), homozygote LL (L group) of fibromyalgia (FM) patients (hatched bars), and genotypes LS and SS (S group) of FM patients (black bars). The results were expressed as mean ± standard error.

Triple-labelling studies using anti-CD3-PE, anti-CD16-FITC and anti-CD56-Cy were applied to analyse NK cells. Only the S group presented significantly reduced levels of mature CD3−CD16+CD56+ NK cells when compared with matched controls (P = 0·04). No significant alteration was observed in the immature CD3−CD16+CD56− NK cells. Analysis of the total CD56+CD3− NK cells revealed the unique significant difference observed between the S and L groups, with decreased levels in the former (P = 0·05) (Fig. 3).

Fig. 3.

Fig. 3

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Absolute numbers of CD56+CD3− natural killer (NK) cells, CD16+CD56+CD3− mature NK cells and CD16+CD56−CD3− immature NK cells on peripheral blood of controls (grey bars), homozygote LL (L group) of fibromyalgia (FM) patients (hatched bars), and genotypes LS and SS (S group) of FM patients (black bars). The results were expressed as mean ± standard error.

Activated lymphocytes

The levels of activated lymphocytes were analysed by the expression of CD25, CD69 and HLA-DR on T lymphocytes, and also of CD23 on B lymphocytes. [31,32]. Significant alterations were detected in the numbers of CD4+ T lymphocytes expressing the activation antigens CD25 and HLA-DR when FM groups were compared individually with matched controls (Table 2). The L group presented increased numbers of CD4+ T lymphocytes expressing the early activation marker CD25 (P = 0·03), but normal levels of CD4+ T cells positive for the late activation antigen HLA-DR. In contrast, patients in the S group had normal levels of CD4+CD25low cells, but increased numbers of CD4+HLA-DR+ lymphocytes (P = 0·04). No remarkable differences were observed in the expression of activation antigens by CD8+ T lymphocytes or CD19+ B lymphocytes.

Table 2.

Absolute numbers of activated T and B lymphocytes.

Control Cells/mm3
L S
CD4+HLA-DR 34 ± 4 45 ± 14 65 ± 17*
CD4+CD25low 112 ± 21 382 ± 110* 207 ± 58
CD4+CD69+ 58 ± 23 43 ± 16 112 ± 55
CD8+HLA-DR 37 ± 10 67 ± 23 54 ± 14
CD8+CD25low 35 ± 18 73 ± 25 62 ± 18
CD8+CD69+ 46 ± 13 42 ± 10 49 ± 14
CD19+CD23+ 48 ± 7 57 ± 9 77 ± 18
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*

P < 0·05. All variables were expressed as mean ± standard error. L group: homozygote LL (L group); S group: genotypes LS and SS. HLA-DR: human leucocyte antigen D-related.

Measuring of cortisol levels

To evaluate the HPA axis function we measured salivary cortisol at three time-points during the day: 8 a.m., 5 p.m. and 10 p.m. Our data showed a significant decrease in salivary cortisol at 8 a.m. in patients of the L group (P = 0·03). In addition, 66% and 75% of patients in the L group presented reduced levels of cortisol at 5 p.m. and 10 p.m. respectively. Concurrently, 46% of patients in the S group presented low levels of cortisol at 8 a.m. (Fig. 4). There was no significant difference between patients and the control group at other time-points.

Fig. 4.

Fig. 4

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Salivary cortisol levels at 8 a.m., 5 p.m. and 10 p.m. in controls (grey bars), homozygote LL (L group) of fibromyalgia (FM) patients (hatched bars), and genotypes LS and SS (S group) of FM patients (black bars). The results were expressed as mean ± standard error.

Discussion

The main purpose of this study was to investigate if the grouping of patients, according to genetic typing, could help in the analysis of immune cells, considering the great discrepancy of data in the literature. We genotyped the promoter region of the serotonin transporter (5-HTT) and defined two groups of FM patients as: the L group, with patients presenting the LL genotype, and the S group, with patients who had at least one short allele (S). We observed increased frequency of the short allele in the patient group, corroborating previous data in the literature [27,28]. A psychiatric interview also showed that the S group had a major depression diagnosis more frequently than the L group, as well as higher scores of anxiety, as evaluated by the STAI questionnaire.

In all case–control genetic studies, we must be aware of false-positive and false-negative findings due to ethnic stratification. Our sample comprised only self-assigned Caucasian–Brazilian individuals. However, as demonstrated recently, race as determined by self- and/or clinical evaluation is a poor predictor of ancestry in Brazil and an ethnic stratification bias cannot be ruled out [33].

We also demonstrated that, although both groups of patients presented hypocortisolism at 8 a.m., this feature was more pronounced in the L group. The association of FM with hypocortisolism has been described by many others [11–15,34], even though some researchers revealed a normal pattern or increased cortisol levels in FM patients [17,18]. Hypocortisolism has been associated with many clinical features of FM, such as fatigue, disturbed sleep, depressive state, stress sensitivity and increased SNS activity [14,35].

Both groups of FM patients were also analysed for counting peripheral blood leucocytes and lymphocyte immunophenotypying, and some relevant data were observed. Compared with healthy controls, patients in the L group had marginally significantly increased numbers of neutrophils, while no significant alteration was observed in the S group. In addition, the L group also presented high numbers of CD4+CD25low activated T lymphocytes. These data indicate disturbance of the innate immune system and systemic early activation of T cells. At this point, it is important to emphasize that the L group also presented hypocortisolism, which could explain in part their increased levels of CD4+CD25low activated T lymphocytes [36,37]. However, correlation analyses between cortisol measuring and CD4+CD25low activated T lymphocytes revealed a weak negative correlation (r = −0·35; P = 0·12) (data not shown). Of note is the recent finding that some FM patients present increased l-selectin expression on neutrophils and monocytes, which suggests increased trafficking of circulating cells into tissues [38].

Patients in the S group also presented signs of systemic immune activation. However, in contrast to the L group they had normal levels of CD4+CD25low, but increased numbers of CD4+ expressing HLA-DR, a marker of late cellular activation. It is plausible to consider that discrepant data in the literature of CD4+CD25low quantification in FM patients [4,8] might be due to different balances of these two genetic groups in those studies, as those studies considered FM patients as a unique homogeneous population [4,8].

Another relevant observation of this study was related to the levels of NK cells. Only patients from the S group had significantly altered levels of CD16+CD56+CD3− NK cells, a phenotype that has been considered to be characteristic of mature NK cells. Some studies in the literature have provided evidence that altered function of NK cells may be associated with depression [39–42]. In this context it interesting to note that, according to clinical analyses, the S group presented a higher prevalence of depression and increased levels of total CD56+CD3− NK cells when compared with the L group.

Also, the two genetic groups presented increased levels of both CD19+CD5− B2 lymphocytes and CD19+CD5+ B1 lymphocytes, and significantly decreased numbers of CD4+CD25high T lymphocytes. The CD4+CD25high phenotype is usually associated in humans with regulatory T lymphocytes, but functional studies and the presence of a set of phenotypic markers are necessary to confirm the regulatory role of these cells in the patients we studied. Interestingly, these features found in FM groups [elevated numbers of B cells and decreased numbers of T regulatory (Treg) cells] are associated commonly with autoimmune diseases [43,44]. In animal models, depletion of Tregs leads to the development of autoimmune diseases in genetically susceptible animals [45]. Furthermore, transfer of Tregs prevents the development of organ-specific autoimmunity, and has been shown to control allograft rejection [46,47] and colitis [48]. Increased levels of CD19+CD5+ B1 lymphocytes are associated commonly with many other autoimmune diseases, such as rheumatoid arthritis and lupus erythematosus, but to our knowledge it has not been described previously in FM patients [43].

In conclusion, our data indicate that FM may be associated with the systemic activation of T lymphocytes, and with autoimmune features. Future follow-up studies are warranted to answer whether such immunological changes play a pathogenic role in FM patients or if they are simply a consequence of a pain-induced neuroimmunological stress response. Further, we provide evidence that genotyping of the promoter region of the serotonin transporter could help to identify FM individuals with differentially altered frequencies of immune cells, which probably justifies the miscellany of results in the literature. More studies, conducted in larger number of patients, are needed to clarify the role of genetics in the diversity of clinical forms presented by FM patients.

Conflicts of interest

The authors have no conflicts of interest concerning the work reported in this paper. We are grateful to Dr Olindo Assis Martins-Filho for statiscal support. This work was supported by CNPQ, Brazil.

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