Chronic fatigue syndrome is associated with diminished intracellular perforin

K J Maher, N G Klimas, M A Fletcher, K J Maher, N G Klimas, M A Fletcher

Abstract

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.

Figures

Fig. 1
Fig. 1
Flow cytometry indicted that the percentage of lymphocytes [T cells and natural killer (NK) cells] co-expressing surface CD2 and CD26 was significantly elevated in the chronic fatigue syndrome (CFS) patients compared to controls. The results are shown as a box–whisker plot. The horizontal lines mark the minimum and maximum, and the 10th, 25th, 50th, 75th and 90th percentile points.
Fig. 2
Fig. 2
Flow cytometry indicated that the number of CD2+ lymphocytes [T cells and natural killer (NK) cells]/µl of blood expressing surface CD26 was elevated in the chronic fatigue syndrome (CFS) patients compared to controls. The results are shown as a box–whisker plot. The horizontal lines mark the minimum and maximum, and the 10th, 25th, 50th, 75th and 90th percentile points.
Fig. 3
Fig. 3
Subjects with chronic fatigue syndrome had significantly lower natural killer (NK) cytotoxicity (NKCC) as determined by the 51Cr release assay, when compared to healthy controls. The results are expressed as a percentage of target cells (K562 cells) killed in the 4 h assay at an effector cell (CD56+CD3− lymphocytes) to target cell ratio of 1 : 1. Results are shown as a box–whisker plot. The horizontal lines mark the minimum and maximum, and the 10th, 25th, 50th, 75th and 90th percentile points.
Fig. 4
Fig. 4
Subjects with chronic fatigue syndrome had significantly lower intracellular perforin in natural killer (NK) cells as determined by quantitative fluorescence. Results are expressed as relative number of molecules of perforin (rMol P) per CD56+ CD3− lymphocyte. The results are shown as a box–whisker plot. The horizontal lines mark the minimum and maximum, and the 10th, 25th, 50th, 75th and 90th percentile points.
Fig. 5
Fig. 5
Representative histograms showing the fluorescence intensity of the antiperforin antibody binding in natural killer (NK) cells The x-axis corresponds to channel number and the y-axis corresponds to relative frequency of events. (a) Healthy control 222641, median fluorescence intensity = 13·2; rMoP per NK cell = 9348. (b) Chronic fatigue syndrome (CFS) patient 234467, median fluorescence intensity = 3·7; rMol per NK cell = 3404.
Fig. 6
Fig. 6
Subjects with chronic fatigue syndrome had a lower intracellular cytotoxic T cell perforin level as determined by quantitative fluorescence that bordered on statistical significance. Results are expressed as relative number of molecules of perforin (rMolP) per CD8+ CD3+ lymphocyte. The results are shown as a box–whisker plot. The horizontal lines mark the minimum and maximum, and the 10th, 25th, 50th, 75th and 90th percentile points.
Fig. 7
Fig. 7
There was a significant correlation between natural killer (NK) cell activity (NKCC) and amount of intracellular perforin (rMolP). Filled symbols represent the individual CFS subjects and control subjects in this study. The line represents the regression line.

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