Potential Role of STAT1 in the Pathogenesis of Chronic Fatigue Syndrome
Konstance K. Knox,
Ph.D. and Donald R. Carrigan,
1Institute for Viral Pathogenesis; 10437 Innovation Drive; Suite 417; Milwaukee, Wisconsin 53226
Project funded by: The National CFIDS Foundation; Needham, Massachusetts
Chronic fatigue syndrome (CFS) is a debilitating illness associated with persistent severe fatigue and a variety of physical and neuropsychological signs and symptoms. While the syndrome itself has been recognized for many years, its etiology and pathogenesis are poorly understood. One of the most intriguing and potentially important aspects of CFS is the unusual susceptibility of individuals with it to a variety of infections. Increased incidences of infections with a variety of viruses (e.g. human herpesvirus six, Epstein-Barr virus and
enteroviruses) and bacteria (e.g. mycoplasma and
chlamydia).1 While this unusual susceptibility to infections is suggestive of an impaired immune system, no single, consistent immune defect is observed although a wide variety of deficiencies has been
Recently advances in the understanding of intracellular signal transduction pathways may have provided a key insight into the immunological defect that may be operative in CFS. Signal transducers and activators of transcription (STAT) are a family of transcription factors that play central roles in the responses of cells to cytokines, molecules that control every aspect of the immune system. Specifically STAT1 and STAT2 are intimately involved in the response of cells to type I (alpha and beta) and type II (gamma)
interferons.2 Genetic defects in STAT1 are associated with fatal infections by both viruses and
The possible importance of STAT1 in CFS stems from recent observations by a number of research groups. First, as reviewed by
Komaroff,6 the type I interferon response is abnormal in patients with CFS. In healthy individuals type
I interferon leads to an intracellular antiviral state that is mediated by the enzyme RNAse L which normally has a molecular weight of 80 kiloDaltons (kDa). However, in patients with CFS the RNAse L induced by type I interferon is abnormally cleaved into a form of only 37 KDa in weight. The protease responsible for the abnormal cleavage in unknown, but it is likely to be closely related to human leukocyte elastase
(HLE).7 In work by
Englebienne9,10 and others, it has been found that patients with CFS express the 37 kDa form of RNAse L in varying degrees and that this variation can be expressed by the ratio of the 37 KDA to 80 kDa forms of the enzyme (termed the "RNAse L ratio"). In a creative set of studies by Englebienne, Fremont et
al9,10 the RNAse L ratios of a set of CFS patients were compared with respect to the expression of STAT1 in the patients peripheral blood mononuclear cells (PBMC).
Remarkably, as the RNAse L ratios increases (higher levels of the abnormal 37 kDa enzyme) the expression of STAT1 protein decreases. When the analysis is performed in the presence of protease inhibitors, this effect is not seen, suggesting the STAT1 protein is being proteolytically
degraded.10 It was proposed that the STAT1 protein is degraded by the same protease responsible for cleavage of the 80 kDa form of RNAse
If these observations and hypothesis prove to be true, the implications for the pathogenesis of CFS would be of great significance. The loss of STAT1's signal transduction function would explain the increased susceptibility of CFS patients to infections and could account for the increased serum levels of interferons that is seen in some patients with CFS. The increased interferon levels would result from the homeostatic increased production of interferon in the face of decreased interferon responsiveness.
Specific Aims of the Proposed Project
The goals of the proposed studies are to confirm and extend the results of Englebienne, Fremont et al. Specifically, we will:
- use monoclonal antibodies and antisera reactive with only the alpha form of STAT1 and with both alpha and beta splice variants of the protein to analyze samples of PBMC from CFS patients and healthy controls in order to determine which of the splice variants is involved
- use monoclonal antibodies specific for the phosphorylated, active form of STAT1 to analyze samples of PBMC from CFS patients and healthy controls
- use both Western Blot and immunocytochemistry with the above antibodies to analyze samples of PBMC from CFS patients and healthy controls
- publish the results that we obtain in an appropriate peer-reviewed journal
Acid citrate dextrose anticoagulated blood samples (5 to 10 milliliters each) will be obtained from 25 patients with CFS and from 15 healthy control individuals. Upon receipt in the laboratory, two ml of blood from each subject will be centrifuged to obtain a plasma sample which is frozen at
-70oC for future studies (e.g. specific serologies or PCR for infectious agents). The remainder of each blood sample will be subjected to density gradient centrifugation using Ficoll-Paque to purify PBMC. After thorough washing in phosphate buffered saline (PBS), the PBMC will be distributed among the following uses:
- cell spots for immunocytochemical staining using multiwell microscope slides obtained from CEL-LINE; Newfield, New Jersey. Prior to immunocytochemical staining the cell spots will be dehydrated in absolute ethanol and fixed in cold
- spun into a cell pellet for processing for denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using the buffer system of Laemmli and Western Blot analysis.
The cell spots and Western Blots will be analyzed by means of three immunologic reagents. All will be purchased from Santa Cruz Biotechnology; Santa Cruz, California. These reagents are:
- STAT1 p84/p91 (M-22): sc-592
This is a rabbit polyclonal antiserum reactive with both splicing variants of STAT 1, i.e. STAT1A and STAT1B. Any staining observed with this reagent in either immunocytochemical and Western Blot procedures will be confirmed to be specific by use of a "blocking
peptide," i.e. the reagent will be incubated with
its specific antigenic target prior to exposure to the patient sample.
- p-STAT1 (A-2): sc-8394
This is a murine monoclonal antibody specific for the phosphorylated tyrosine-701 (Tyr-701) of both STAT1A and STAT1 B. It does not react with the unphosphorylated forms of either splice variant. As with the sc-592 reagent, any staining observed with this reagent in either immunocytochemical and Western Blot procedures will be confirmed to be specific by use of a "blocking
- STAT1a p91 (C-111): sc-417
This is a murine monoclonal antibody specific for the alpha splicing variant of STAT1; it does not recognize the beta splicing variant.
This reagent was the sole antibody used in the work of Englebienne, Fremont et
al.9,10 There is no "blocking peptide" available for this reagent.
In both the immunocytochemical and Western Blot procedures, antibodies bound to antigen will be detected by means of the appropriate horseradish peroxidase (HRP) labeled second antibody (specific for either rabbit or murine IgG) in combination with diaminobenzidine (DAB) as an enzyme substrate.
- De Meirleir K, De Becker P, Nijs J et al. CFS etiology, the immune system and infection. In Chronic Fatigue Syndrome: A Biological Approach. Eds. P Englebienne and K De Meirleir. CRC Press; New York (2002). pps.201-228.
- Stark GR, Kerr IM, Williams BRG, Silverman RH and Schreiber. How cells respond to interferons. Ann Rev Biochem 1998; 67:227-264.
- Dupuis S, Jouanguy E, Al-Hajjar S et al. Impaired response to interferon-a/b and lethal viral disease in human STAT1 deficiency. Nat Genet 2003;
- Durbin JE, Hackenmiller R, Simon MC and Levy DE. Targeted disruption of the mouse STAT1 gene results in compromised innate immunity to viral diseases.
- Wang J, Schreiber RD and Campbell. STAT1 deficiency unexpectedly and markedly exacerbates the pathophysiological actions of IFN-a in the central nervous system. Proc Natl Acad Sci 2002; 99:16209-16214.
- Komaroff AL. The biology of chronic fatigue syndrome. Amer J Med. 2000; 108:169-171.
- Demettre E, Bastide L, D'Haese et al. Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients. J Biol Chem 2002; 38:35746-35751.
- Suhadolnik RJ, Reichenbach NL, Hitzges P et al. Upregulation of the 2-5 synthetase/RNase l antiviral pathway associated with chronic fatigue syndrome. Clin Infect Dis 1994; S96-S104.
- Englebienne P, Herst CV, Fremont M et al. The 2-5A pathway and signal transduction: a possible link to immune dysregulation and fatigue. In Chronic Fatigue Syndrome: A Biological Approach. Eds. P Englebienne and K De Meirleir. CRC Press; New York (2002). pps.99-130.
- Fremont M, Englebienne P and Herst CVT. Methods for diagnosis and treatment of chronic immune diseases. United States Patent Application Publication Number US2003/0077674 A1. April 24, 2003.