Unique immunologic patterns in fibromyalgia

  • Frederick G Behm1,

    Affiliated with

    • Igor M Gavin2,

      Affiliated with

      • Oleksiy Karpenko2,

        Affiliated with

        • Valerie Lindgren1,

          Affiliated with

          • Sujata Gaitonde1,

            Affiliated with

            • Peter A Gashkoff1 and

              Affiliated with

              • Bruce S Gillis1Email author

                Affiliated with

                BMC Clinical Pathology201212:25

                DOI: 10.1186/1472-6890-12-25

                Received: 8 July 2012

                Accepted: 10 December 2012

                Published: 17 December 2012

                Abstract

                Background

                Fibromyalgia (FM) is a clinical syndrome characterized by chronic pain and allodynia. The diagnosis of FM has been one of exclusion as a test to confirm the diagnosis is lacking. Recent data highlight the role of the immune system in FM. Aberrant expressions of immune mediators, such as cytokines, have been linked to the pathogenesis and traits of FM. We therefore determined whether cytokine production by immune cells is altered in FM patients by comparing the cellular responses to mitogenic activators of stimulated blood mononuclear cells of a large number of patients with FM to those of healthy matched individuals.

                Methods

                Plasma and peripheral blood mononuclear cells (PBMC) were collected from 110 patients with the clinical diagnosis of FM and 91 healthy donors. Parallel samples of PBMC were cultured overnight in medium alone or in the presence of mitogenic activators; PHA or PMA in combination with ionomycin. The cytokine concentrations of IFN-γ, IL-5, IL-6, IL-8, IL-10, MIP-1β , MCP-1, and MIP1-α in plasma as well as in cultured supernatants were determined using a multiplex immunoassay using bead array technology.

                Results

                Cytokine levels of stimulated PBMC cultures of healthy control subjects were significantly increased as compared to matched non-stimulated PBMC cultures. In contrast, the concentrations of most cytokines were lower in stimulated samples from patients with FM compared to controls. The decreases of cytokine concentrations in patients samples ranged from 1.5-fold for MIP-1β to 10.2-fold for IL-6 in PHA challenges. In PMA challenges, we observed 1.8 to 4-fold decreases in the concentrations of cytokines in patient samples.

                Conclusion

                The cytokine responses to mitogenic activators of PBMC isolated from patients with FM were significantly lower than those of healthy individuals, implying that cell-mediated immunity is impaired in FM patients. This novel cytokine assay reveals unique and valuable immunologic traits, which, when combined with clinical patterns, can offer a diagnostic methodology in FM.

                Background

                Fibromyalgia (FM) is a chronic pain and fatigue syndrome that affects ten to fifteen million adults in the United States. Patients with FM display diffuse hyperalgesia (i.e., increased response to painful stimuli) and/or allodynia (i.e., a heightened sensitivity to stimuli that are not normally painful). Other clinical manifestations include debilitating fatigue, sleep disturbance, joint stiffness, cognitive dysfunction and depression. In the past, this syndrome has been known as fibrositis, nonarticular rheumatism, the chronic fatigue syndrome, myodysneuria, fibromyositis and muscular rheumatism, among others. The diagnosis of FM is one of exclusion as there are no specific laboratory tests or radiographic/imaging studies to confirm this diagnosis. The only physical finding currently used for diagnosis is excess tenderness on palpation of soft tissues [15]. Patients who have been labeled with the diagnosis of FM commonly spend years seeking confirmation of this health problem. The lack of an objective test to either confirm or rule-out the existence of FM not infrequently results in some patients being labeled as neurotic, hypochondrical or emotionally unstable.

                The etiology of FM is not completely understood but the current hypothesis is that FM arises from interactions between the autonomic central nervous system, the hypothalamic-pituitary-adrenal axis and the immune system [69]. In the past, FM was considered a rheumatologic disorder based upon the assumption that it is a type of inflammatory fibro-connective malady. This is not surprising given that FM is common in patients with autoimmune disorders, such as systemic lupus erythematosus [10, 11], Sjogren's Syndrome [12, 13], and rheumatoid arthritis [14]. Recent studies have highlighted the role of the immune system in the pathogenesis of this disease [15]. As early as 1988, it was noted that aberrant expressions of immune mediators such as cytokines may contribute to the onset of disease symptoms [16]. However, reports on changes in serum cytokine levels in FM patients have revealed conflicting results [17, 18]. To uncover direct evidence of the imbalance in cytokine production and secretion in FM patients we evaluated the cytokine-producing activity of immune cells in FM patients. We measured plasma cytokine levels in a group of 110 patients with a diagnosis of FM and we determined responses to mitogen challenges of their peripheral blood mononuclear cells (PBMC). The cytokine levels of these patients were compared with those in a group of 91 matched healthy controls.

                Methods

                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

                Cohort size

                10

                Age

                 Range

                18-82

                 Median

                53.2

                 Mean

                52.2

                 Sex

                 

                 Female

                98

                 Male

                12

                Ethnicity

                 Caucasian*

                101

                 Other

                9

                Years of living with FM

                 Range

                1-46

                 Median

                30

                 Mean

                16

                 Mode

                10

                Fibromyalgia Symptoms/Co-Morbidities

                 Tender Muscle Points

                101

                 Nonrestorative Sleep/Insomnia

                89

                 Chronic Fatigue

                99

                 Mental Fogginess

                93

                 Psychotherapist Confirmed Diagnosis of Depression

                9

                * The Caucasian classification was also used if patients identified themselves as Hispanic.

                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% K3 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 CO2 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].

                Results

                Concentrations of cytokines in plasma of FM patients

                Concentrations of eight cytokines, IFN-γ, IL-5, IL-6, IL-8, IL-10, MIP-1β, MCP-1, and MIP1-α were measured in the plasma of the 110 FM patients and the 91 control subjects (Table 2). The levels of most cytokines were below the lower limits of detection. When cytokine levels in FM samples were compared with the control values, no statistically significant differences were found at α=0.05.
                Table 2

                Concentrations of cytokines in plasma of FM patients and controls

                Cytokine

                Controls

                Patients

                P-value

                N

                Mean

                SD

                N

                Mean

                SD

                MCP-1

                65

                25

                19

                95

                23

                23

                0.5681

                IL-5

                30

                24.0

                86.0

                26

                28

                103

                0.8685

                IL-6

                39

                23

                59

                47

                18

                29

                0.6511

                IL-8

                62

                22

                13

                43

                25

                49

                0.7031

                IL-10

                59

                125

                713

                54

                18

                41

                0.2531

                IFN-γ

                54

                659

                1935

                51

                681

                766

                0.9383

                MIP-1α

                36

                6

                7

                24

                9

                24

                0.6807

                MIP-1β

                87

                61

                31

                107

                52

                49

                0.1506

                Concentrations of cytokines in pg/ml are shown.

                N, number of subjects whose cytokine values within the detection ranges.

                Levels of cytokines in stimulated PBMC cultures

                To determine if cytokine production by immune cells is altered in FM patients, we measured cellular responses to the mitogenic activators, phytohemagglutinin (PHA) and phorbol-12-myristate-13-acetate (PMA) in combination with ionomycin. PHA, a lectin from Phaseolus vulgaris (red kidney bean), induces PBMC proliferation as well as the secretion of cytokines [21, 22]. PMA and ionomycin also stimulate PBMC and activate extracellular expressions of cytokines [2325]. We therefore cultured PBMC which were isolated from the blood samples from the 110 patients and the 91 control subjects in the presence of either PHA or PMA/ionomycin and determined the concentrations of eight cytokines in culture supernatants. As shown in Table 3, unstimulated PBMC cultures produced low levels of cytokines in the control subjects. Of these 91 tested samples, about half were below the lower detection limits. In contrast, in both of the PHA and PMA/ionomycin challenges, extracellular expressions of all cytokines were significantly increased, resulting in increased cytokine levels in the culture supernatants. The only exception was the cytokine IL-5 concentrations in the PHA challenge, whose increase was not statistically significant.
                Table 3

                Concentrations of cytokines in stimulated PBMC cultures of controls

                Cytokine

                Median detection ranges

                Unstimulated

                PHA-stimulated

                PMA/Ionomycin-stimulated

                N

                Mean

                SD

                N

                Mean

                SD

                P-value

                N

                Mean

                SD

                P-value

                MCP-1

                3-14,000

                50

                299

                217

                90

                3497

                4384

                <.001

                91

                1224

                1126

                <.001

                IL-5

                1-13,000

                16

                2.6

                1.3

                32

                48

                150

                0.10

                87

                64

                64

                <.001

                IL-6

                1-33,000

                59

                215

                380

                91

                2799

                4182

                <.001

                91

                532

                474

                <.001

                IL-8

                2-60,000

                56

                771

                659

                89

                17456

                24246

                <.001

                90

                17607

                10103

                <.001

                IL-10

                2-63,000

                44

                23

                29

                65

                80

                94

                <.001

                87

                63

                60

                <.001

                IFN-γ

                5-224,000

                40

                538

                338

                70

                1516

                2425

                0.001

                91

                36726

                55772

                <.001

                MIP-1α

                1-7,000

                56

                33

                73

                91

                1084

                1773

                <.001

                90

                1246

                1051

                <.001

                MIP-1β

                5-14,000

                57

                222

                174

                83

                4413

                5228

                <.001

                80

                6389

                4915

                <.001

                Concentrations of cytokines in pg/ml are shown.

                N, number of subjects whose cytokine values were within the detection ranges.

                PBMC responses to mitogens in FM patients versus controls

                When cytokine concentrations in patient samples were compared to the control values, the concentrations of most cytokines were lower in FM samples (Table 4). The decrease was statistically significant for all cytokines except for IL-5 in the PHA challenge. Besides muscle tenderness, the vast majority of the FM patients suffered from chronic fatigue, sleep disorders as well as mental fogginess, which is consistent with the universally recognized traits that make up the FM syndrome. Therefore, the samples from the subgroup of patients having the same individual traits had a nearly identical change in cytokine values when compared to the whole group (not shown). Nine FM patients had been diagnosed by a licensed psychotherapist with depression. A statistical analysis was performed on the patients excluding those who had a confirmed diagnosis of depression and it showed similar results (not shown), thereby proving that depression by itself had an extremely limited impact on the cytokine profiles in FM patients.
                Table 4

                Cytokine production in response to stimulation in FM patients compared to healthy controls

                Cytokine

                PHA

                Controls

                Patients

                Cmean/ Pmean

                P-value

                N

                Mean

                SD

                Min.

                Max.

                Med.

                N

                Mean

                SD

                Min.

                Max.

                Med.

                MCP-1

                90

                3497

                4384

                69

                >OOR

                1823

                109

                1968

                1993

                86

                >OOR

                1310

                1.8

                0.003

                IL-5

                32

                47.7

                150.1

                <OOR

                853

                14.1

                48

                7

                17

                <OOR

                89

                1.5

                7

                0.136

                IL-6

                91

                2799

                4182

                1.2

                15592

                273

                110

                276

                437

                3.1

                2255

                108

                10.2

                <.001

                IL-8

                89

                17456

                24246

                161

                >OOR

                4933

                110

                5751

                6123

                135

                37981

                3144

                3

                <.001

                IL-10

                65

                80

                94

                <OOR

                352

                38

                103

                12

                15

                <OOR

                84

                6.8

                6.5

                <.001

                IFNγ

                70

                1516

                2425

                <OOR

                16902

                670

                101

                569

                1920

                <OOR

                15318

                124

                2.7

                0.007

                MIP-1α

                91

                1084

                1773

                1.9

                9159

                240

                110

                204

                272

                5.7

                1598

                109

                5.3

                <.001

                MIP-1β

                83

                4413

                5228

                122

                >OOR

                2282

                109

                2900

                2417

                395

                >OOR

                2259

                1.5

                0.016

                Cytokine

                PMA/Ionomycin

                Controls

                Patients

                C mean / P mean

                P- value

                N

                Mean

                SD

                Min.

                Max.

                Med.

                N

                Mean

                SD

                Min.

                Max.

                Med.

                MCP-1

                91

                1224

                1126

                150

                8295

                874

                108

                436

                421

                <OOR

                2363

                292

                2.8

                <.001

                IL-5

                87

                63.8

                63.9

                <OOR

                374.5

                42.5

                98

                36

                54

                <OOR

                441

                17

                1.8

                0.002

                IL-6

                91

                532

                474

                26

                2108

                350

                108

                242

                301

                <OOR

                1509

                109

                2.2

                <.001

                IL-8

                90

                17607

                10103

                5724

                >OOR

                13677

                109

                8756

                6416

                215

                >OOR

                7503

                2

                <.001

                IL-10

                87

                63

                60

                <OOR

                254

                40

                90

                27

                37

                <OOR

                230

                11

                2.4

                <.001

                IFNγ

                91

                36726

                55772

                205

                339591

                16130

                103

                14584

                23047

                <OOR

                144516

                5636

                2.5

                0.001

                MIP-1α

                90

                1246

                1051

                105

                >OOR

                987

                109

                308

                294

                <OOR

                1551

                191

                4

                <.001

                MIP-1β

                80

                6389

                4915

                254

                >OOR

                5177

                106

                3086

                4040

                9.5

                >OOR

                1517

                2.1

                <.001

                Concentrations of cytokines in pg/ml are shown.

                N, number of subjects whose cytokine values were within the detection ranges.

                OOR, out of range.

                In order to better understand the dynamics of changes in individual cytokines in patients, we questioned whether expressions of different cytokines changed similarly in the same samples. We calculated pair-wise correlations between the cytokine concentrations using Spearman's rank correlation (Table 5). All of the cytokines showed moderate to strong correlations with each other with a correlation coefficient ρ > 0.4 (all correlations were significant at 1% FDR), suggesting that cytokine dynamics after each treatment is similar in individual patients. No negative correlations were observed, which means that no two cytokine expressions changed in opposite directions.
                Table 5

                Spearman correlation coefficients of cytokine expressions in patients

                PHA

                MCP-1

                IL-5

                IL-6

                IL-8

                IL-10

                IFNγ

                MIP-1α

                MIP-1β

                MCP-1

                1

                0.52

                0.68

                0.80

                0.69

                0.41

                0.61

                0.71

                IL-5

                 

                1

                0.67

                0.68

                0.69

                0.65

                0.76

                0.66

                IL-6

                  

                1

                0.82

                0.82

                0.76

                0.90

                0.80

                IL-8

                   

                1

                0.74

                0.67

                0.84

                0.71

                IL-10

                    

                1

                0.57

                0.85

                0.82

                IFNγ

                     

                1

                0.78

                0.55

                MIP-1α

                      

                1

                0.82

                MIP-1β

                       

                1

                PMA

                MCP-1

                IL-5

                IL-6

                IL-8

                IL-10

                IFNγ

                MIP-1α

                MIP-1β

                MCP-1

                1

                0.60

                0.64

                0.77

                0.52

                0.49

                0.67

                0.51

                IL-5

                 

                1

                0.74

                0.48

                0.67

                0.72

                0.71

                0.69

                IL-6

                  

                1

                0.57

                0.83

                0.83

                0.88

                0.83

                IL-8

                   

                1

                0.48

                0.39

                0.63

                0.43

                IL-10

                    

                1

                0.86

                0.86

                0.85

                IFNγ

                     

                1

                0.85

                0.93

                MIP-1α

                      

                1

                0.87

                MIP-1β

                       

                1

                Discussion

                We utilized multiple immunologic methods to develop an objective test that offers an adjunct to the current subjective physical findings which are used to make a diagnosis when a patient is suspected of having FM. This test is based on specific abnormalities in the cytokine levels of stimulated peripheral blood mononuclear cells of such patients. Decreased PBMC responses to challenges regarding IFN-γ, IL-5, IL-6, IL-8, IL-10, MIP-1β, MCP-1 and MIP1-α but not IL-5 were statistically significantly associated with this disorder in a large number of patients with FM. Decreased expressions of IL-6, IL-10, and IFN-γ were also observed for purified protein derivative-stimulated PBMC in patients with Sjogren’s syndrome with myalgia [26]. The fibromyalgia-related cytokine patterns which were identified may lend some insight as to why there has been an overlap in the diagnoses of mental depression and chronic pain in these individuals. Chronically depressed patients are characterized by higher pro-inflammatory cytokine blood levels and lower anti-inflammatory cytokine levels in blood, similar to what has been found in patients with chronic pain [2731]. Decreased IL-10, IL-1α and IFN-γ production by mitogen-stimulated PBMC was observed in depressed patients which correlated with our findings [32, 33].

                The methodology used in this study differs significantly from previous studies; therefore the findings of this study cannot be directly compared to the results of previous studies of cytokines in patients with FM. For example, many studies examined levels of cytokines in serum or plasma, used different controls or did not ensure that their patients were not receiving treatments for FM [17, 18]. Our findings prove that relying on a methodology that solely measures circulating cytokine levels leads to results that are statistically insignificant and such measurements fail to confer any diagnostic values.

                There have been several studies which employed isolated peripheral blood cells which were stimulated with different agents and they measured levels of a limited number of cytokines after stimulation [3437]. The latter studies further differed from the methods used in this study regarding the types of PBMCs used (e.g., whole blood, isolated lymphocytes and monocytes alone or together) and in the methods of measurement (e.g., ELISA, flow cytometry, intracellular cytokine staining, and RT-PCR). Regardless of the methodology used, our study revealed significant differences in cytokine expressions in PBMCs from FM patients versus control cells, which can be utilized as the basis of a clinical diagnostic test for FM.

                The common thread in these studies as related to the current study was a reduction or no increase in the levels of cytokines IL-6, IL-8, IL-10 and IFN-γ in patients with chronic pain.

                The fibromyalgia syndrome by definition lacks any consistent patterns regarding pain intensity, which is a totally subjective process. Pain duration is a required criteria per the American College of Rheumatology definition for the diagnosis of FM, which we adhered to. In the past, FM was claimed to be a rheumatologic, neurologic or psychiatric disease despite the fact that there were no objective links to any of those pathways. Our findings uncovered evidence that FM is instead an immunologic disorder. They prove that the immunologic basis of FM occurs independently of any subjective features. Hence, this illustrates the very strong clinical value of our test protocol. The fact that individual cytokines exhibited similar dynamics in patient samples reveals that the FM patients are uniform in regard to their cellular immunologic responses.

                Conclusion

                We conclude that the aberrant responses of levels of a large number of cytokines of in vitro stimulated PBMC using the methodology in this study is significantly correlated with the clinical diagnosis of FM. This methodology provides a useful confirmation of the clinical diagnosis of FM. Further, this method of evaluating cytokines levels in stimulated PBMC may prove useful in analyzing specific responses to therapeutic modalities and medications in order to determine the efficacy of such interventions.

                Declarations

                Authors’ Affiliations

                (1)
                Department of Pathology, University of Illinois at Chicago (UIC)
                (2)
                Research Resource Center, University of Illinois at Chicago

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                38. Pre-publication history

                  1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1472-6890/​12/​25/​prepub

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