ERK1/2, MEK1/2 and p38 downstream signalling molecules impaired in CD56 dim CD16+ and CD56 bright CD16 dim/- natural killer cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients

Abstract

Background: Natural Killer (NK) cell effector functions are dependent on phosphorylation of the mitogen-activated protein kinases (MAPK) pathway to produce an effective immune response for the clearance of target cells infected with viruses, bacteria or malignantly transformed cells. Intracellular signals activating NK cell cytokine production and cytotoxic activity are propagated through protein phosphorylation of MAPKs including MEK1/2, ERK1/2, p38 and JNK. Reduced NK cell cytotoxic activity is consistently reported in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) patients and intracellular signalling by MAPK in NK cells remains to be investigated. Therefore, the purpose of this paper was to investigate MAPK downstream signalling molecules in NK cell phenotypes from CFS/ME patients.

Methods: Flow cytometric protocols were used to measure phosphorylation of the MAPK pathway in CD56(bright)CD16(dim/-) and CD56(dim)CD16(+) NK cells following stimulation with K562 tumour cells or phorbol-12-myristate-13-acetate plus ionomycin. NK cell cytotoxic activity, degranulation, lytic proteins and cytokine production were also measured as markers for CD56(bright)CD16(dim/-) and CD56(dim)CD16(+) NK cell function using flow cytometric protocols.

Results: CFS/ME patients (n = 14) had a significant decrease in ERK1/2 in CD56(dim)CD16(+) NK cells compared to the non-fatigued controls (n = 11) after incubation with K562 cells. CD56(bright)CD16(dim/-) NK cells from CFS/ME patients had a significant increase in MEK1/2 and p38 following incubation with K562 cells.

Conclusions: This is the first study to report significant differences in MAPK intracellular signalling molecules in CD56(dim)CD16(+) and CD56(bright)CD16(dim/-) NK cells from CFS/ME patients. The current results highlight the importance of intracellular signalling through the MAPK pathway for synergistic effector function of CD56(dim)CD16(+) and CD56(bright)CD16(dim/-) NK cells to ensure efficient clearance of target cells. In CFS/ME patients, dysfunctional MAPK signalling may contribute to reduced NK cell cytotoxic activity.

Keywords: Chronic Fatigue Syndrome/Myalgic Encephalomyelitis; Cytokine; Cytotoxic; Intracellular signalling; Mitogen-activated protein kinase signalling; Natural killer.

Fig. 1

Measurement of the ERK1/2 in CD56^dimCD16⁺ NK cells. For a representative individual, CD56^dimCD16⁺ NK cells were identified according to the surface expression of CD56 and CD16 (a) which was extrapolated onto a histogram plot to identify median fluorescence intensity (MFI) of ERK1/2 in US, K562 and PMA/I stimulated cells (b). Compared to the NFC, phosphorylated ERK1/2 in CD56^dimCD16⁺ NK cells from CFS/ME patients was significantly reduced (c, *p < 0.05). Data are presented as MFI with interquartile range

Fig. 2

MEK1/2 and p38 measurement in CD56^brightCD16^dim/− NK cells. For a representative individual, CD56^brightCD16^dim/− NK cells were identified using CD56 and CD16 (a). CD56^brightCD16^dim/− NK cell MEK1/2 was determined on a histogram (b) and comparisons were drawn between CFS/ME and the NFC under different stimulatory conditions (c). In comparison to the NFC, MEK1/2 was significantly increased in CFS/ME patients after K562 cell incubation (*p < 0.05). p38 MFI in CD56^brightCD16^dim/− NK cells was also measured on histograms (d) and comparisons were drawn against the CFS/ME patients and the NFC (e) before and after stimulation. In CFS/ME patients, p38 was significantly increased (*p < 0.05) after K562 incubation compared to the NFC. Data are presented as MFI with interquartile range