PETER H. SCHUR,4 PHILLIP K. PETERSON,2 and ANTHONY L. KOMAROFF1 ' ... major depression, systemic lupus erythematosis (SLE), and multiple sclerosis ...
Journal of Clinical Immunology, Vol. 17, No. 2, 1997
Elevation of Bioactive Transforming Growth Factor-j3 in Serum from Patients with Chronic Fatigue Syndrome ADRIENNE L. BENNETT,1 CHUN C. CHAO,2 SHUXIAN HU,2 DEDRA BUCHWALD,3 LAURA R. FAGIOLI,' PETER H. SCHUR,4 PHILLIP K. PETERSON,2 and ANTHONY L. KOMAROFF1 '
Accepted: November 18, 1996
Chronic fatigue syndrome (CFS) is an idiopathic illness characterized by disabling fatigue and a constellation of somatic and neuropsychological symptoms (1, 2). Although the etiology and pathogenesis of CFS are unknown, a variety of immunological abnormalities has been described (3, 4), suggesting that the immune system could play a pathogenic role. Several proinflammatory cytokines, including interferon a (IFNa), interleukin-1 (IL-1), and tumor necrosis factor a (TNFa), are known to induce symptoms such as fatigue and somnolence similar to those reported by CFS patients (5, 6). The possibility that cytokines are involved in the pathogenesis of CFS is supported by studies demonstrating increased levels of IL-1 a (7), interleukin-2 (IL-2) (8), neopterin (9), interleukin-6 (IL-6) (9), IFNa (10), and tumor necrosis factors-a and -j3 ( 1 1 ) in the serum or plasma of CFS patients compared to normal individuals. However, normal levels of IFNa (7, 12, 13), TNFa (12, 13), and IL-2 (12) have been found in other studies of CFS. In two small studies of CFS patients living in Minnesota, another cytokine, transforming growth factor-/3 (TGF-/3) was found to be elevated in the serum of the CFS patients compared to healthy control subjects (14, 15). The purpose of the present study was to extend these observations regarding serum TGF-/3 levels to a larger group of CFS patients from another geographicarea and to compare them to several disease comparison groups in which pathologic fatigue and/or immunologic abnormalities are prominent features.
The level of bioactive transforming growth factor-/} (TGF-/3) was measured in serum from patients with chronic fatigue syndrome (CFS), healthy control subjects, and patients with major depression, systemic lupus erythematosis (SLE), and multiple sclerosis (MS) of both the relapsing/remitting (R/R) and the chronic progressive (CP) types. Patients with CFS had significantly higher levels of bioactive TGF-/3 levels compared to the healthy control, major depression, SLE, R/R MS, and CP MS groups (P < 0.01). Additionally, no significant differences were found between the healthy control subjects and any of the disease comparison groups. The current rinding that TGF-/3 is significantly elevated among patients with CFS supports the findings of two previous studies examining smaller numbers of CFS patients. In conclusion, TGF-/3 levels were significantly higher in CFS patients compared to patients with various diseases known to be associated with immunologic abnormalities and/or pathologic fatigue. These findings raise interesting questions about the possible role of TGF-j3 in the pathogenesis of CFS. KEY WORDS: Transforming growth factor-/3; chronic fatigue syndrome; major depression; systemic lupus erythematosis; multiple sclerosis.
1 Chronic Fatigue Syndrome Cooperative Research Center and Division of General Medicine and Primary Care, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115. 2 Department of Medicine, Hennepin County Medical Center, Neuroimmunobiology and Host Defense Laboratory, Minneapolis Medical Research Center, and University of Minnesota Medical School, Minneapolis, Minnesota 55415. 3 Department of Medicine, University of Washington, Seattle, Washington 98102. 4 Lupus Center, Division of Rheumatology and Immunology, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115. 5 To whom correspondence should be addressed at Division of General Medicine and Primary Care, Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115.
MATERIALS AND METHODS Subject Selection Chronic Fatigue Syndrome Patient Group. This group consisted of 93 patients, all meeting the original 1988 160 (l271-9142/97/0300-0160$12.50/0
© 1997 Plenum Publishing Corporation
TGF-J3 IN CHRONIC FATIGUE SYNDROMK
Centers for Disease Control criteria for the diagnosis of CFS that was operative at the time the study was conducted (1). The patients were recruited from the CFS Cooperative Research Center based at Brigham and Women's Hospital (BWH). In addition, all patients reported the symptom of postexertional malaise. Five CFS patients were dropped from the study: three had serum samples which were toxic to the HT-2 cell cultures, and two had an insufficient quantity of serum for testing. Healthy Subject Group. This control group consisted of 80 healthy volunteers, as determined by self-report, matched for age and gender to the CFS group. Of these, 54 were blood donors who explicitly denied various current illnesses, and 26 were either patients seen for routine health care at a primary care practice or hospital personnel. Four healthy control subjects were dropped from the study: three had serum samples which were toxic to the HT-2 cell cultures, and one had an insufficient quantity of serum for testing. Major Depression Disease Comparison Group. This group included a total of 46 patients with unipolar major depression. Of these, 21 were not on antidepressant medication and lived in or near Seattle, WA. Twenty-five were patients taking antidepressant medication and resided in the greater Boston, MA, area. Two depressed patients subsequently were dropped from the study: one had a manic episode, and therefore a diagnosis of bipolar disorder, and another because the serum could not be tested due to technical difficulties. The symptom of fatigue was not routinely assessed in this group. Systemic Lupus Erythematosis (SLE) Disease Comparison Group. This group consisted of 50 patients in varying stages of disease activity, who were followed at the Arthritis Center at Brigham and Women's Hospital. Many were on immunosuppressive therapy: 17 were taking a steroid, and of this group, 5 reported the symptom of fatigue; 10 were taking both a steroid and imuran, and of this group, 2 reported the symptom of fatigue; 3 were taking imuran alone, and of this group, 1 reported the symptom of fatigue; and I was taking a steroid and cytoxan, and this patient did not report fatigue. Information regarding medications and fatigue was not available for 4 of the 50 patients. Multiple Sclerosis (MS) Disease Comparison Groups. One group included 41 MS patients with the relapsing/ remitting form of MS (designated R/R MS), were in remission, and had not received steroids within the past month prior to sample collection. The other group of 16 MS patients had the chronic progressive form of MS (designated CP MS), and all had active disease, but none had been on steroids within the past month prior to
Journal of Clinical Immunology, Vol. 17, No. 2, 1997
sample collection. All MS patients were recruited from an MS Clinic at BWH. The symptom of fatigue was not routinely assessed in this group. Serum Sample Preparation All testing was performed on coded specimens; for all specimens the laboratory was blind to information regarding the subjects' group status. Samples were aliquoted and frozen at -20°C and were shipped on dry ice in six shipments over the course of approximately 1 year. Each shipment contained samples from several of the six patient groups. TGF-P Assay TGF-/3 was measured using a bioassay and previously reported methods (14). This bioassay uses murine HT-2 cell cultures in which TGF-/31 (porcine; R&D Systems, Inc., Minneapolis, MN) specifically inhibits IL-4dependent cell proliferation. Briefly, HT-2 cells maintained in RPMI 1640 containing 10% FCS and IL-2 (35 ng/ml) were seeded onto 96-well plates at a concentration of 104 cells per well in RPMI 1640 medium containing 10% FCS and IL-4 (15 ng/ml) in the absence of IL-2. Heat-inactivated serum samples (50 /xl) or recombinant human TGF-/31 in a series of dilutions was added to each well and incubated for 48 hr. Next, 50 JJL\ of 3-[4,5-dimethylthiazol-Z-ylJ-2,5-diphenyltetrazolium bromide, thiazolyl blue (5 mg/ml; Sigma), was added, and the plates were incubated overnight prior to being read at a 550-nm wavelength. The ED5D of the TGF-)3 assay was 15 ± 1 pg/ml (n = 20) and the sensitivity was 1 PgTo confirm the specificity of TGF-/3 in serum samples, anti-TGF-j8 was added to over 90% of the specimens tested in this study. Polyclonal rabbit antibodies to TGF-01 (10 jug/ml) provided by R&D Systems blocked >50% of TGF^ bioactivity in these serum samples. Minor variation (