Authors

Kennedy G, Spence VA, Underwood C, Belch JJF

Institution

Vascular Diseases Research Unit, The Institute of Cardiovascular Research, Ninewells Hospital and Medical School, Dundee, UK

Support

The study was funded by ME Research UK, with further support received from the Sir John Fisher Foundation(Educational Grant).

Introduction

Many patients with chronic fatigue syndrome (CFS) have symptoms that are consistent with an underlying viral or toxic illness. Considering that increased neutrophil apoptosis occurs in patients with infection, we examined whether patients with CFS also demonstrate increased numbers of neutrophils undergoing apoptosis.

Methods

Forty-seven patients (18 M & 29F, mean age 47.5 years [19–63 years]) who fulfilled the Centers for Disease Control classification for CFS were enrolled into the study. Thirty-four sex- and aged-matched healthy volunteers (13 M & 21F, 45.9 years [19-63 years]) were also recruited. The apoptotic process was measured isolating the neutrophils and incubating them with annexing V and or propidium iodide and by measuring leukocyte TNF-RI surface expression levels using a fluorescent-labeled antibody. An ELISA was used to measure platelet poor plasma levels of TGF-b1.

Results

Patients with CFS had significantly higher levels of neutrophil annexing V and TNF-RI expression (37.4 vs 22.8%, p=0.001 [Mann-Whitney U-test]; and 12.5 vs 3.9%, p=0.004; respectively) and increased plasma levels of TGF-b1 (2.24 vs 1.89 pg/mL, p=0.005) when compared with controls. The patient group had significantly lower number of viable neutrophils (63.0 vs 77.0%, p=0.002) and an increased number of early apoptotic neutrophils (32.0 vs 19.6%, p=0.002).

Conclusions

We have shown that patients with CFS also have increased neutrophil apoptosis and higher levels of TGF-b1. We suggest that increased neutrophil apoptosis and inhibition of transmigration of neutrophils by higher TGF-b1 levels may be indicative of a persistent viral infection or a toxic state giving rise to many of the symptoms which characterise CFS. It might also be that increased apoptosis merely reflects a quicker turnover of neutrophils in this condition, but either way the data presented here provides convincing evidence that many patients with CFS have an underlying detectable abnormality.

Publication

Journal of Clinical Pathology 2004 Aug; 57(8): 891–3

Presentation

International Society on Thrombosis and Haemostasis, Birmingham, July, 2003. Journal of Thrombosis and Haemostasis, 2003 Jul 12–18; 1(Suppl 1): P1258

Published comments

Non-technical summary

Apoptosis is the programmed destruction of unwanted cells in the body. It is an important process which removes cells that have reached the end of their natural life, as well as controlling infections. Apoptosis is carried out by white blood cells called neutrophils, which are part of the immune system.

From measuring levels of various chemical markers in their blood, we found that patients with ME/CFS had abnormally high rates of apoptosis, as well as a lower number of functioning neutrophils, than healthy people of the same age.

These findings may have been caused either by a persistent viral infection or by a quicker than normal turnover of neutrophils. Either way, they represent a significant, detectable abnormality in the immune system of patients with ME/CFS.

Comment by ME Research UK

Some of the symptoms of ME/CFS are suggestive of an underlying viral or toxic illness associated with persistent infection and immune activation. Indeed, there have been various reports of immunological disturbances and viral infections in the illness (1). Neutrophils represent 50–60% of the total circulating white blood cells, and are short-lived reactive cells fundamental to the functioning of an intact immune system. Minor alterations to neutrophil function can have profound immunological consequences, with amplification of the inflammatory response and the production of cytokines. As part of the resolution of inflammation, accumulated neutrophils are removed by apoptosis, a process where unwanted or damaged cells are eliminated without releasing their toxic contents and enhancing the inflammatory response. It is also a process associated with release of anti-inflammatory mediators, most specifically the production of transforming growth factor beta1 (TGF-b1), implicated in the pathogenesis of CFS.

While increased neutrophil apoptosis is present in patients with infection (2), this is the first time elements of neutrophil function have been determined in ME/CFS patients. The importance of these findings lies in the fact that neutrophils from ME/CFS patients have a greater proportion of apoptotic cells and are significantly less viable when compared with healthy subjects. The same neutrophils expressed a higher percentage of the death receptor TNF-RI and had increased binding of annexin V, indicative of phosphatidylserine exposure. Apoptosis is triggered by signals initiated by both external stimuli and internal sensors. The death receptor mediated pathway or the extrinsic pathway starts with the binding of TNF-family ligands to the death receptor TNF-RI, and results in a cascade of immune events. There is, however, crosstalk between the extrinsic and the intrinsic (mitochondrial-dependent) pathways which can result in the release of cytochrome C and the triggering of further apoptotic mechanisms These authors have demonstrated that ME/CFS patients have an increased neutrophil expression of TNF-RI, and surmise that the accelerated apoptosis of these cells is a consequence of extrinsic factors affecting apoptotic pathways. The additional finding that neutrophils from ME/CFS patients have more surface expression of TNF-RI further indicates that such cells are obviously more susceptible to apoptosis.

An increased rate of apoptosis of neutrophils may impact on the innate immune system of ME/CFS patients given that neutrophils are the major effector cells of this system. The decision for neutrophils to undergo accelerated apoptosis is a complex one and, in these patients, it may be a consequence of several factors. Accelerated apoptosis is indicative of a persistent or reactivating infection or a toxic state, reprogramming of apoptotic pathways by an infectious or toxic agent, or quicker neutrophil turnover, secondary to an abnormal host response to noxious stimuli.

With the advent of gene profiling, the search is on for causative agents in ME/CFS. In combination with previous reports of increased apoptosis in lymphocytes (rather than neutrophils) in these patients (3, 4), the data presented by these authors is consistent with the presence of an underlying, detectable abnormality in the immune cell behaviour of ME/CFS patients, consistent with an activated inflammatory process.

The trustees would like to acknowledge the support of the Corporate Friends of ME Research UK which enables this and other biomedical research studies to be undertaken.

References

  1. Patarca R, Mark T, Fletcher MA et al. Review: Immunology of Chronic Fatigue Syndrome. J Chronic Fatigue 2000; 6: 69–107.
  2. Nwakoby IE, Reddy K, Patel P et al. Fas-mediated apoptosis of neutrophils in sera of patients with infection. Infect Immun 2001; 69: 3343–9.
  3. Hassan IS, Bannister BA, Akbar A et al. A study of the immunology of the chronic fatigue syndrome: correlation of immunologic parameters to health dysfunction. Clin Immunol Immunopathol 1998; 87: 60–7.
  4. Vojdani A, Ghoneum M, Choppa PC et al. Elevated apoptotic cell population in patients with chronic fatigue syndrome: the pivotal role of protein kinase RNA. J Intern Med 1997; 242: 465–78.