Summary:
Chronic fatigue syndrome (CFS) is an illness characterized by unexplained and prolonged fatigue that is often accompanied by abnormalities of immune, endocrine and cognitive functions. Diminished natural killer cell cytotoxicity (NKCC) is a frequently reported finding. However, the molecular basis of this defect of in vitro cytotoxicy has not been described.

Perforin is a protein found within intracellular granules of NK and cytotoxic T cells and is a key factor in the lytic processes mediated by these cells. Quantitative fluorescence flow cytometry was used to the intracellular perforin content in CFS subjects and healthy controls. A significant reduction in the NK cell associated perforin levels in samples from CFS patients, compared to healthy controls, was observed. There was also an indication of a reduced perforin level within the cytotoxic T cells of CFS subjects, providing the first evidence, to our knowledge, to suggest a T cell associated cytotoxic deficit in CFS.

Because perforin is important in immune surveillance and homeostasis of the immune system, its deficiency may prove to be an important factor in the pathogenesis of CFS and its analysis may prove useful as a biomarker in the study of CFS.

[ ... ]

Discussion:
Many of the symptoms of CFS are inflammatory in nature (myalgia, arthralgia, sore throat, tender lymphadenopathy), and have prompted a theory of infection induced illness. CFS often presents with acute onset of illness (reported in 60–80% of published samples) with systemic symptoms similar to influenza infection that do not subside [13]. However, reports of associated microbial infection or of latent virus reactivation have been inconsistent. Whether associated with a known antigen (e.g. a specific infection) or not, there is a considerable literature describing immune activation in CFS. These reports have described the elevation of lymphocyte surface activation markers [6,14], the expression of proinflammatory cytokines and evidence of Th2 cytokine increase [15–17]. In order to determine if the cohort of CFS patients in this study had evidence of immune activation we elected to analyse the surface marker, CD26 (dipeptidyl peptidase IV). This ectoenzyme is known to increase upon cell activation [18]. DPPIV/CD26 is a multifunctional molecule that is a proteolytic enzyme, receptor, co-stimulatory protein, and is involved in adhesion and apoptosis. CD26 is associated on T cells with adenosine deaminase (ADA), and plays a major role in immune response. Abnormal expression is found in autoimmune diseases, HIV-related diseases and cancer [19]. Compared to controls, the CFS patients we studied had a significantly elevated percentage and absolute count of CD26+ lymphocytes.

The laboratory finding reported with the greatest consistency in CFS patients is that of reduced NKCC [7,9]. In the few studies that failed to find depressed NK activity in CFS subjects, methodological problems may have been responsible [7]. In the present study, consistent with previous reports, the NK-mediated cytotoxicity of CFS subjects against the K562 cell targets was significantly lower than that of controls. Although the CFS subjects in this cohort also had significantly lower numbers of NK cells (CD56+ CD3) per volume of blood, the diminished cytotoxicity was due to a decreased functional capacity of the NK cells, as the percentage of killing in the 51Cr release assay was calculated for both subject groups at a 1 : 1 target to CD56+ CD3 effector cell ratio. NK cells are critical for immune surveillance against fungal, bacterial and viral infections. They also play a vital role in cellular resistance to malignancy and tumour metastasis [20]. NK cells can destroy their target cells by calcium-dependent release of cytolytic granules, by activation of the Fas (CD95) pathway or through contact with tumour necrosis factor (TNF)-α[21]. Perforin is released along with granzymes, particularly granzyme B, from intracellular vesicles of cytolytic effector cells and facilitates passage of these molecules through target cell membranes, which then activate the apoptotic pathways of the caspases [21]. NK cells differ from the other cytotoxic effector cell types (CTL) in two major ways: they kill the target cells in a non-major histocompatibility complex (MHC)-restricted fashion without the need for previous in vitro or in vivo activation, and only NK cells express constitutively the lytic machinery [20,21].

There was a recent suggestion in the literature of perforin reduction in CFS. Steinhau et al. [22] used differential display polymerase chain reaction (PCR) to search for candidate biomarkers in CFS. RNA expression profiles of one subject with CFS and an age- and sex-matched control showed differential expression of 10 genes. Of these, five were down-regulated and one was perforin. In the present report, we found that the relative number of molecules of perforin per NK cells from CFS patients was significantly below that found in matched healthy controls. This finding added support to the concept of an NK-associated immune deficit in CFS and suggested that the decrease in cytolytic potential of NK cells might be associated with a reduction in the cell-associated concentrations of the effector molecule perforin. A similar finding, which approached statistical significance (P = 0·06), was a deficit in the perforin content of the cytotoxic T subset. The observed decrease of perforin in T cells was of an even greater magnitude than that seen in NK cells (~30% and 55% of control levels for Tc and NK, respectively). To our knowledge, this is the first report of evidence to suggest a deficit in the cytotoxic T cell compartment of CFS patients. These findings have considerable significance in providing a potential mechanism in the pathogenesis of CFS.

Two areas of research provided important insight in the role of perforin in normal and pathological conditions. The perforin knockout mouse, a model of perforin deficiency, demonstrated the role of perforin as a cytotoxic effector molecule through decreased cytotoxicity against virus infected and allogeneic targets [23] and reduced clearance of intracellular pathogens and tumours [24,25]. These mice had increased numbers of activated CD8 cells [26] and altered cytokine production with elevated expression of interleukin (IL)-1, interferon (IFN)-γ and TNF-α[27–29]. In the absence of perforin, the granule protein granzyme A was proinflammatory, and induced IL-6 and IL-8 expression [30].

A second area of investigation that pointed to the significance of perforin in health and disease comes from studies of humans with the condition known as familial haemophagic lymphohistiocytosis (FHL). This rare and fatal disorder of early childhood was associated with a genetic mutation that, in the homozygous state, resulted in the absence of perforin expression [31]. Individuals with FHL were found to suffer from an extensive immune activation, expansion and infiltration of activated lymphocytes throughout the body, increased expression of proinflammatory cytokines (IFN-γ, TNF-α, IL-1 and IL-6) and severely impaired cytotoxic abilities [32–34].

Given the deficiency of perforin among patients with CFS reported here and the role of perforin in immune surveillance and immunomodulation, we suggest that decreased intracellular perforin content may play a role in the pathogenesis of CFS, an illness reported to be associated with increased immune activation, inflammatory cytokine levels, herpes virus reactivation and decreased cytotoxicity. The mechanism responsible for the reduced perforin in CFS remains unknown. The mechanisms may include genetic deficiency or chronic microbial activation leading to perforin consumption and exhaustion. An example of the latter mechanism is found in the report of reduced NKCC and reduced intracellular perforin in patients with chronic hepatitis C virus infection [35]. CFS is an illness whose symptomatic expression is variable and any hypothosis regarding the development of CFS should account for this variability. It can be envisioned that either mechanism mentioned above could be subject to variability. The diversity of symptoms may be due to the degree of perforin deficiency, as a gene dosage effect is seen in mice [36] and humans [34]; those who are heterozygous for the deficiency display an intermediate phenotype. Secondly, exposure to activating stimuli may be necessary for the evolution of symptomatic CFS in that perforin deficiency in both children and C57BL/6 perforin knockout mice is not associated with pathology until a viral infection stimulates an uncontrolled immune activation [36,37]. Thus, superimposition of immune activation, either microbial, autoimmune or even allergic, may vary between those susceptible and alter the course of illness. Thirdly, lymphocytes have multiple mechanisms to mediate killing. The perforin pathway of cytotoxicity is distinct from that mediated by Fas or TNF-α. Perforin is essential for both lytic and granzyme-mediated apoptotic killing but not Fas-mediated killing [21]. Because these systems provide a similar function, varying levels of redundancy among patients may explain varying degrees of susceptibility to developing symptomatic illness.

Conclusions:
We have presented evidence of a significant reduction in the intracellular content of perforin among patients with chronic fatigue syndrome. This molecule plays an important role in immune surveillance against microbes and neoplasia as well as in immune homeostasis. Such a deficiency is likely to be associated with altered immune function and we would propose that this finding may be important in the pathogenesis of CFS. However, this pathogenic process is probably multifactorial, and variations in susceptibility and inciting stimuli may account for the constellation of symptoms seen in CFS. Perforin deficiency may prove useful as a biological marker in CFS − perhaps one that will help define a subgroup with a common pathogenesis.

Acknowledgements:
This work was funded by support from the NIH Center Grant 1UD 1-AI 45940, the Miami Veterans Affairs Research and Education Foundation and the CFIDS Association of America.

References:
1 Fukuda K, Straus SE, Hickie I et al. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med 1994; 121: 953–9.
2 Reyes M, Nisenbaum R, Hoaglin DC et al. Prevalence and incidence of chronic fatigue syndrome in Wichita, Kansas. Arch Intern Med 2003; 163: 1530–6.
3 Jason LA, Richman JA, Rademaker AW et al. A community-based study of chronic fatigue syndrome. Arch Intern Med 1999; 159: 2129–37.
4 Reyes M, Dobbins JG, Nisenbaum R, Subedar NS, Randall B, Reeves WC. Chronic fatigue syndrome progression and self-defined recovery: evidence from the CDC surveillance system. J Chronic Fatigue Syndrome 1999; 5: 17–27.
5 Hill NF, Tiersky LA, Scavalla VR, Lavietes M, Natelson BH. Natural history of severe chronic fatigue syndrome. Arch Phys Med Rehabil 1999; 80: 1090–4.
6 Klimas NG, Salvato FR, Morgan R, Fletcher MA. Immunologic abnormalities in chronic fatigue syndrome. J Clin Microbiol 1990; 28: 1403–10.
7 Maher K, Klimas NG, Fletcher MA. Immunology. In: Jason L, Fennell P, Taylor R, eds. Handbook of chronic fatigue syndrome. Hoboken, NJ: John Wiley and Sons, 2003: 124–51.
8 Caligiuri M, Murray C, Buchwald D et al. Phenotypic and functional deficiency of natural killer cells in patients with chronic fatigue syndrome. J Immunol 1987; 139: 3306–13.
9 Fletcher MA, Maher K, Klimas NG. Natural killer cell function in chronic fatigue syndrome. Clin Appl Immunol Rev 2002; 2: 129–39.
10 Centers for Disease Control and Prevention (CDC). 1997 revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR 1997; 46: 1–29.
11 Maher KJ, Klimas NG, Hurwitz B, Schiff R, Fletcher MA. Quantitative fluorescence measures for the determination of intracellular perforin content. Clin Diagn Lab Immunol 2002; 9: 1248–52. 12 Patarca R, Fletcher MA, Podack E. Cytotoxic lymphocytes. In: Rose N, deMacario E, Folds J, Lane HC, Nakamura R, eds. Manual of clinical laboratory immunology, 5th edn. Washington, DC: ASM Press, 1997: 29–303.
13 Plioplys A. Differential diagnosis in medical assessment. In: Jason L, Fennell P, Taylor R, eds. Handbook of chronic fatigue syndrome. Hoboken, NJ: John Wiley and Sons, 2003: 26–41.
14 Landay AL, Jessop C, Lennette ET, Levy JA. Chronic fatigue syndrome: clinical condition associated with immune activation. Lancet 1991; 338: 707–12.
15 Skowera A, Cleare A, Blair D, Bevis L, Wessely SC, Peakman M. High levels of type 2 cytokine-producing cells in chronic fatigue syndrome. Clin Exp Immunol 2004; 135: 294–302.
16 Gupta S, Aggarwal S, See D, Starr A. Cytokine production by adherent and non-adherent mononuclear cells in chronic fatigue syndrome. J Psych Res 1997; 31: 149–56.
17 Patarca R, Klimas NG, Lugtendorf S et al. Dysregulated expression of tumor necrosis factor in chronic fatigue syndrome: interrelations with cellular sources and patterns of soluble immune mediator expression. Clin Infect Dis 1994; 18: S147–53.
18 Kameoka J, Tanaka T, Nojima Y, et al. Direct association of adenocine diaminase with T cell activation antigen, CD26. Science 1994; 261: 466–70.
19 Boonacker E, Van Noorden CJ. The multifunctional or moonlighting protein CD26/DPPIV. Eur J Cell Biol 2003; 82: 53–73.
20 Moretta L, Bottino D, Pende MC et al. Human natural killer cells: their origin, receptors and function. Eur J Immunol 2002; 32: 1205–11.
21 Barry M, Bleackley R. Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol 2002; 2: 401–9.
22 Steinau M, Unger ER, Vernon SD et al. Differential-display PCR of peripheral blood for biomarker discovery in chronic fatigue syndrome. J Mol Med 2004; 82: 750–5.
23 Kagi D, Ledermann B, Burki K et al. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 1994; 369: 31–7.
24 Kagi D, Ledermann B, Burki K et al. CD8+ T cell-mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity. Eur J Immunol 1994; 24: 3068–72.
25 Tang Y, Hugin AW, Gies NA et al. Control of immunodeficiency and lymphoproliferation in mouse AIDS: studies of mice deficient in CD8+ T cells or perforin. J Virol 1997; 71: 1808–13.
26 Kagi D, Odermatt B, Mak TW. Homeostatic regulation of CD8+ T cells by perforin. Eur J Immunol 1999; 29: 3262–72.
27 Sambhara S, Switzer I, Kurichh A et al. Enhanced antibody and cytokine responses to influenza viral antigens in perforin-deficient mice. Cell Immunol 1998; 187: 13–18.
28 Kehren J, Desvignes C, Krasteva M et al. Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity. J Exp Med 1999; 189: 779–86.
29 Binder D, van den Broek MF, Kagi D et al. Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus. J Exp Med 1998; 187: 1903–20.
30 Sower LE, Klimpel GR, Hanna W et al. Extracellular activities of human granzymes. I. Granzyme A induces IL6 and IL8 production in fibroblast and epithelial cell lines. Cell Immunol 1996; 171: 159–63.
31 Stepp SE, Dufourcq-Lagelouse R, Le Deist F et al. Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science 1999; 286: 1957–9.
32 Henter JI, Elinder G, Soder O et al. Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991; 78: 2918–22.
33 Arico M, Janka G, Fischer A et al. Hemophagocytic lymphohistiocytosis. Report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society. Leukemia 1996; 10: 197–203.
34 Sullivan KE, Delaar CA, Douglas S et al. Defective natural killer cell function in patients with hemophagocytic lymphohistiocytosis and in first degree relatives. Pediatr Res 1998; 44: 465–8.
35 Par G, Rukavina D, Podack E et al. Decrease in CD3-negative-CD8dim and Vδ2/Vγ9 TcR+ peripheral blood lymphocyte counts, low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection. J Hepatol 2002; 37: 514–22.
36 Matloubian M, Suresh M, Glass A et al. A role for perforin in downregulating T-cell responses during chronic viral infection. J Virol 1999; 73: 2527–36.
37 Henter JI, Ehrnst A, Andersson J et al. Familial hemophagocytic lymphohistiocytosis and viral infections. Acta Pediatr 1993; 82: 369–72.