Clinical relevance of antibodies against serotonin and gangliosides in patients with primary fibromyalgia syndrome

Psychon~uroendocrinology, Vol. 17. No. 6. pp. 593-598, 1992

0306-4530/92 S5.00-~0.00 ©1993 Pergamon Press Ltd.

Printed in Great Britain

CLINICAL RELEVANCE OF ANTIBODIES AGAINST SEROTONIN AND GANGLIOSIDES IN PATIENTS WITH PRIMARY FIBROMYALGIA SYNDROME REINHILD KLEIN, MATTHIAS B.~NSCH, and PETER A. BERG Department of Internal Medicine, University of Ti~bingen. Tiibingen, Germany (Received 19 June 1991; in final form 16 September 1991)

SUMMARY The fibromyalgia syndrome (FMS) is a non-articular rheumatic disorder associated with disturbances in serotonin metabolism. In order to evaluate whether patients with FMS suffer from an autommune disorder, we tested sera from 50 clinically well-defined FMS patients for non-organspecific and organ-specific antibodies by enzyme-linked immunosorbent assay and immunofluorescence test. Common antibodies against nuclei, mitochondria, and microsomes were not increased in these patients compared to healthy controls. However, 74% had antibodies against serotonin and gangliosides. The clinical and diagnostic relevance of these antibodies is supported by the absence of anti-serotonin antibodies in other rheumatic disorders such as rheumatoid arthritis, polymyalgia rheumatica, and collagen diseases. These antibodies may belong to the group of antireceptor antibodies, considering the fact that gangliosides are an important component of the serotonin receptor. It remains to be determined whether these antibodies are of pathogenetic relevance, interfering with serotonin binding and thereby inducing symptoms associated with FMS.

INTRODUCTION THE PRIMARY FIBROMYALGIAS Y N D R O M E (FIVIS) is a form of non-articular rheumatism. Muscle pain is the most important symptom, and tender points at multiple characteristic sites can be demonstrated in most instances (Yunus et al., 1981). It is frequently associated with chronic fatigue, poor sleep, irritable bowel, headaches, depression, and dysmenorrhea (Bennett, 1989; Yunus et al., 1989). All series confirm the female preponderance as well as the chronicity of symptoms in this syndrome (Wolfe & Cathey, 1983; Goldenberg, 1987; Yunus et al., 1989). The differential diagnosis from other rheumatic diseases may be difficult, and a defined pathophysiological basis is lacking. Muscle biopsies reveal no inflammatory processes (Kalyanraman et al., 1984), and biochemical parameters are normal in most patients. Psychological profiles of patients with FMS revealed elevated scores for depression, hypochondriasis, and hysteria, and current or past psychiatric disturbance has been reported in up to 72% of patients (Wolfe et al., 1985; Goldenberg, 1989a; Merskey, 1989; Yunus et al., 1989). Primary FMS should be differentiated from secondary cases, which are related to an underlying cause (secondary FMS) (Yunus et al., 1989). Address correspondence and reprint requests to: Prof. Dr. P. A. Berg, Department of Internal Medicine, University of Tiibingen, Otfried Miiller Stral]e, D-7400 Tilbingen, GERMANY. 593

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Based on the concept that primary FMS may be related to some immunological alterations, we screened a well-defined group of patients for a large spectrum of antibodies which have been shown to occur in primary or secondary autoimmune diseases. Since some autoimmune disorders also have been related to the induction of anti-hormone and anti-receptor antibodies (Cohen et al., 1984; Strosberg, 1984), we included in this antibody screening the determination of antibodies against gangliosides, which have been shown to be part of the serotonin receptor (Fishman, 1988), as well as against serotonin itself. The latter test was established in light of observations suggesting that alterations of serotonin metabolism may be involved in the etiopathogenesis of FMS (Moldofsky et al., 1975; Goldenberg, 1989b). SUBJECTS A N D M E T H O D S Fifty patients with FMS, all of whom fulfilled the proposed criteria for a diagnosis of fibromyalgia (Yunus et aL, 1981), were included in the study (41 women, 9 men; mean age 45.4 yr, range 2 5 - 6 9 yr). All patients had been seen clinically by one of us (P.A.B.). They had generalized musculoskeletai aching and tender points at multiple muscle sites, in the absence of an underlying condition. Laboratory findings, including complete blood count, erythrocyte sedimentation rate, serum chemistry profile, immunoglobulin levels, and thyroid function test, were normal in all instances. As controls we tested sera from 24 patients with rheumatoid arthritis, 8 patients with polymyalgia rbeumatica, 30 patients with antinuclear antibody-positive collagen disorders, 128 patients with polyneuropathy due to diabetes mellitus, uremia, or alcohol-toxic processes, and 32 blood donors. Antigens used in the enzyme-linked immunosorbent assay (ELISA) Serotonin (5-hydroxytryptamine) and human epidermal keratin were obtained from Sigma (St. Louis MO, USA), gangliosides (Gml, GDla, GDIb, GTIb) from Boehringer (Mannheim, Germany), and laminin from Medac (Hamburg, Germany). Patients' sera also were tested for antibodies to tryptophan (AS), a precursor of serotonin. This preparation was shown to be a pure fraction by high performance liquid chromatography (HPLC) (Belongia et al., 1990). We also used as antigen a fraction from bovine central nervous system tissue ("CNS-specific antigen fraction") (Klein et al., 1991). ELISA Patients' sera were tested by ELISA against the aforementioned antigens by a standard method (Engvall, 1980), as described by Klein and Berg (1988). The optimal antigen concentrations and serum dilutions were determined for each antigen by serial dilutions. Best results were obtained at an antigen concentration of 10 I.tg/ml for each antigen and at serum dilutions of 1:500 for serotonin and 1:1000 for the gangliosides, keratin, laminin, and "CNS-specific fraction". Microtiter plates were incubated with the antigens overnight at 4 ° C. The antigens were diluted in hydrogen carbonate buffer, 0.2 M, pH 9.6. The plates were washed three times with phosphate-buffered saline (F'BS), pH 7.4, containing 1% bovine serum albumin (BSA), and incubated with this buffer for 60 min at room temperature in order to block free binding sites. The buffer then was discarded. Diluted patients' sera were added for 90 min. After a PBS wash containing 0.5% BSA and 0.1% Triton X 100, perioxidase-conjugated anti-human IgG and IgM antibodies (Dianova, Hamburg, Germany) were added in parallel at a dilution of 1:3000. After incubation at room temperature for 60 min, the plates were again washed three times, and o-phenylenediamine, 0.5 mg/ml in 0.2 M citrate buffer, pH 5.0, and 0.01% H202, was added as substrate. The reaction was read at 492 nm in an ELISA® autoreader (Dynatech). In order to exclude nonspecific binding, the patients' sera also were incubated with uncoated plates. As standard sera we used three sera with high, medium, and low titers of antibodies against serotonin, as well as a negative control. Normal values were determined by testing sera from 32 blood donors for anti-serotonin antibodies. The means and standard deviations (SD) of the optical densities (OD) of these sera were calculated. Patients' sera with ODs above the mean+2 SD were defined as borderline cases, and sera with ODs above the mean +3 SD were defined as positive for anti-serotonin antibodies.

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Immunofluorescence test

The patients' sera also were tested for antibodies against cell nuclei (ANA), smooth muscle antigens (SMA), and mitochondria (AMA) by an immunofluorescence test (IFL) performed on cryostat sections from rat liver, heart, kidney, stomach, and human thyroid (Berg et al., 1967). Patients' sera were diluted 1:10 for this test. Determination of serum serotonin levels

Serum serotonin levels were determined with a test kit (Dianova, Hamburg, Germany). It is an immunoassay which uses the competition between acylated serotonin and acetylcholinesterase coupled to serotonin for an antibody fixed to microtiter plates. The normal range in serum is 500-900 nmol/ml. RESULTS The autoantibody frequencies in the 50 patients with FMS are shown in Table I. Patients' sera were tested by IFL for common autoantibodies as well as by ELISA for antibodies against laminin, keratin, and organ-specific antibodies against CNS-tissue. No differences between the FMS patients and healthy controls were observed for the common antigens, except for antithyroid and anti-keratin antibodies. In contrast, sera from patients with FMS reacted significantly with serotonin and gangliosides by ELISA: 37 of the 50 patients (74%) were positive for each of the antigens, and, in most instances, both antibodies were present. Fifteen patients had anti-serotonin antibodies only of the IgG type, nine had antibodies only of the IgM type, and thirteen had antibodies of both types. The same results were obtained when the sera were tested against pure tryptophan. The clinical relevance of this antibody type was highlighted by the testing of sera from 62 patients with other rheumatic disorders for antibodies against serotonin and gangliosides. The overall incidence was 15%, indicating the frequent presence of antibodies against serotonin in FMS (Table II). Antibodies against gangliosides were more frequently found than anti-sero-

TABLE I. A U T O A N T I B O D Y PROFILES IN 50 PATIENTS WITH FIBROMYALGIA S Y N D R O M E (FMS) A N D IN 32 B L O O D D O N O R CONTROLS

FMS Patients Number (%)

Blood Donors Number (%)

Common antibodies Antinuclear antibodies (> 1:20) Smooth muscle antibodies Antimitochondrial antibodies Antithyroid antibodies Anti-CNS antibodies Anti-laminin antibodies Anti-keratin antibodies

3 2 0 7 0 4 7

(6) (4) (14) (8) (14)

0 2 0 1 0 3 2

(6) (3) (9) (6)

Fibromyalgia syndrome-related antibodies Serotonin Gangliosides (Gml, GDla, GDlb, GTlb)

37 (74) 37 (74)

2 (6) 4 (13)

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TABLEII.

SPECIFICITY OF ANT[-SEROTONIN ANTIBODIES FOR THE DIAGNOSIS OF FIBROMYALGIA SYNDROME

Number (%) positive

Number of patients tested

Fibromyalgia syndrome Rheumatoid arthritis Polymyalgia rheumatica Collagen disorders Polyneuropathy

for antibodies to:

Serotonin

Diagnosis

50 24 8 30 128

37 2 2 4 13

(74) (8) (25) (13) (10)

Gangliosides 37 5 2 6 63

(74) (21) (25) (20) (49)

tonin antibodies and occurred preferentially in patients with polyneuropathy. With respect to the two patients with rheumatoid arthritis and the other two patients with polymyalgia rheumatica being anti-serotonin antibody-positive, there was evidence for concomitant fibromyalgia in these patients, as clinically shown by the presence of typical tender points. Serum serotonin levels were determined in all 50 patients with FMS. They were normal in 45 patients; only 5 patients had decreased levels. DISCUSSION This is the first study, to our knowledge, demonstrating diagnostically relevant antibodies in FMS patients. We found antibodies against the neurotransmitter, serotonin, in 37 of 50 patients (74%), and in most instances this antibody was associated with antibodies against gangliosides. Twenty-eight patients had anti-serotonin antibodies of the IgG type. In contrast, only 15% of patients with other rheumatic disorders had this antibody type. The same results were obtained in testing patients' sera against purified tryptophan, which has a very similar molecular structure as serotonin and is a precursor of this neuropeptide. These data indicate that the antibodies may cross-react with these two substances and they exclude a non-specific reaction with a contaminant which could have been present in the serotonin preparation. Considering the frequent association of anti-serotonin and anti-ganglioside antibodies, as well as the fact that gangliosides are components of serotonin receptors (Fishman, 1988), one could speculate that they may be anti-receptor antibodies. Since many symptoms in FMS have been related to lowered serotonin levels (Sicuteri, 1972; Moldofsky et al., 1975; Moldofsky & Scarisbrick, 1975; van Woert & Sethy, 1975; Goldenberg, 1989b), we also measured circulating serotonin in our FMS patients. We were unable to detect a decrease, despite the presence of antibodies against this neurotransmitter. It therefore is likely that blocking of serotonin receptors by these antibodies, and not decreased serotonin levels, may be responsible for the symptoms. The platelets of patients with migraine and depression have significantly decreased serotonin binding (Briley et al., 1980; Paul et al. 1981; Geaney et al., 1984; Meltzer & Arora, 1991), and preliminary data indicate that these patients also have anti-serotonin antibodies (unpublished observations). Since platelets have been considered as a model of 5-hydroxytryptamine neurons (Pletscher, 1968; Sneddon, 1973; Stahl, 1977; Meltzer & Arora, 1991), one can hypothesize that in the brain, as well, abnormal serotonin binding may occur due to the presence of blocking antibodies. In order to compensate this relative serotonin deficiency,

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receptors on platelets (and 5-hydroxytryptamine neurons) may be increased (Russell et al., 1987). The etiology of FMS is still unknown. One of its symptoms, chronic fatigue, can be triggered by a viral infection, e.g., due to Epstein-Barr virus or cytomegalovirus (cf. Komaroff & Goldenberg, 1989, for review). The induction of antiserotonin antibodies also might be the consequence of an infectious process by an agent-exposing epitopes which may be recognized by B-cell clones that have been primed to produce this antibody type. This hypothesis accounts for the well-documented observation that autoantibodies may be derived from the pool of naturally occurring antibodies (Holmberg & Coutinho, 1985; Cohen & Cooke, 1986; Klein & Berg, 1990). Our finding that patients with FMS do not produce any other autoantibody type frequently observed in a large spectrum of rheumatic disorders must be taken as evidence that only a oligoclonal stimulation in the B-cell repertoire occurs. The slight increase of antibodies against keratin and thyroid known to occur during infectious processes and in healthy individuals (Avrameas et al., 1983; Bouanani et al., 1989) also favors the concept of an oligoclonal stimulation of B-cells by cross-reacting epitopes of an infectious agent. Although these immunological data are still difficult to interpret in regard to the various clinical findings in patients with FMS, they do indicate that this rheumatic disorder has a close relationship to alterations of the neuroendocrine regulatory system.

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