Circulating NK cells and their subsets in Behçet's disease

Abstract

Behçet's disease (BD) is an autoinflammatory, chronic relapsing/remitting disease of unknown aetiology with both innate and acquired immune cells implicated in disease pathogenesis. Peripheral blood natural killer (NK) cells and their CD56^Dim /CD56^Bright subsets were surface phenotyped using CD27 and CD16 surface markers in 60 BD patients compared to 60 healthy controls (HCs). Functional potential was assessed by production of interferon (IFN)-γ, granzyme B, perforin and the expression of degranulation marker CD107a. The effects of disease activity (BD^Active versus BD^Quiet ) and BD medication on NK cells were also investigated. Peripheral blood NK cells (P < 0·0001) and their constituent CD56^Dim (P < 0·0001) and CD56^Bright (P = 0·0015) subsets were depleted significantly in BD patients compared to HCs, and especially in those with active disease (BD^Active ) (P < 0·0001). BD patients taking azathioprine also had significantly depleted NK cells compared to HCs (P < 0·0001). A stepwise multivariate linear regression model confirmed BD activity and azathioprine therapy as significant independent predictor variables of peripheral blood NK percentage (P < 0·001). In general, CD56^Dim cells produced more perforin (P < 0·0001) and granzyme B (P < 0·01) expressed higher CD16 levels (P < 0·0001) compared to CD56^Bright cells, confirming their increased cytotoxic potential with overall higher NK cell CD107a expression in BD compared to HCs (P < 0·01). Interestingly, IFN-γ production and CD27 expression were not significantly different between CD56^Dim /CD56^Bright subsets. In conclusion, both BD activity and azathioprine therapy have significant independent depletive effects on the peripheral blood NK cell compartment.

Keywords: Behçet's disease; NK cells; immunopathology; immunotherapies and innate immunity.

Figure 1

Natural killer (NK) cell peripheral blood percentage decreases independently of age in Behçet's disease (BD)^Active patients. (a) NK cells are defined using flow cytometry as CD56⁽⁺⁾ CD3^(–), with two main subsets characterized by intensity of CD56 expression CD56^Bright and CD56^Dim (dot‐plot shown from a single healthy control sample to demonstrate population gating). (b) NK cells CD56⁺CD3^– are expressed as a proportion of total gated lymphocytes in healthy controls (HC) (n = 60) compared to BD patients with no clinical signs of activity BD^Quiet (n = 16) and those with active systems BD^Active patients (n = 44) (total BD; n = 60). (c) NK/gated lymphocyte (%) is compared in both total BD and HC patients across age ranges 15–34, 35–54 and 55–75 years. CD56^Bright NK cells expressed as a proportion of total NK cells is compared between HC and BD patients. (d) Gender differences in NK/gated lymphocyte (%) is compared between HC and BD patients. *P < 0·05; **P < 0·01; ***P < 0·001; n.s. = not significant. Numbers in parentheses above each bar indicate number of individuals in each comparison group.

Figure 2

CD56^Dim and CD56^Bright natural killer (NK) subsets are phenotypically and functionally distinct. (a) Difference in percentage of CD56^Dim (far left) and CD56^Bright subsets (middle) as a percentage of gated lymphocytes with change in CD56^Bright subset expressed as a percentage of total NK cells (far right). (b) Flow cytometry histogram plots showing intensity of surface marker expression of CD27 and CD16 and intracellular cytokine staining for interferon (IFN)‐γ, perforin and granzyme B following 5‐h phorbol myristate actate (PMA)/ionomycin stimulation (representative plots from healthy control sample are shown). (c) Summary median fluorescent intensities (MFI) of gated positive events for CD27, CD16, IFN‐γ, perforin and granzyme B showing relative differences in expression intensities of markers on each of NK subset (CD56^Dim and CD56^Bright) in healthy controls (HC) and in Behçet's disease (BD) patients. *P < 0·05; **P < 0·01; ***P < 0·001; n.s. = not significant.

Figure 3

Comparison of percentage expression of CD27, CD16 and interferon (IFN)‐γ between natural killer (NK)^Dim and NK^Bright subsets. Percentage expression of surface markers CD27 and CD16 alongside intracellular staining for the cytokine IFN‐γ and cytotoxic proteins perforin and granzyme B following 5‐h phorbol myristate acetate (PMA)/ionomycin stimulation for (a) CD56^Dim and (b) CD56^Bright NK subsets. (c) Percentage CD107a expression is shown on total NK cells in heathy control (HC) individuals (n = 6) compared to Behçet's disease (BD) patients (n = 14) following 5‐h PMA/ionomycin activation (left panel). Summary median fluorescent intensities (MFI) of gated positive events are shown for CD107a showing relative differences in expression intensities of markers on each of NK subset (CD56^Dim and CD56^Bright) in HC and in BD patients. *P < 0·05; **P < 0·01; ***P < 0·001; n.s. = not significant.

Figure 4

Natural killer (NK) cell peripheral blood percentage is reduced significantly in patients under azathioprine therapy. (a) NK cells expressed as a percentage of total gated lymphocytes was compared in patients on different forms of Behçet's disease (BD) medication; colchicine (BD^Col), prednisolone (BD^Pred), mycophenolate mofetil (BD^MMF) and azathioprine (BD^Aza) and compared to healthy controls (HC) as well as patients not currently taking BD‐associated medication (BD^{No therapy}). (b) NK cell percentage expressed as a percentage of total lymphocytes in BD patients subgrouped according to disease activity (BD^Quiet/Active) and whether currently taking azathioprine or not (BD^Aza or BD^Non‐Aza). *P < 0·05; **P < 0·001; ***P < 0·001. Aza = azathioprine; Col = colchicine; MMF = mycophenolate mofetil; Pred = prednisolone; Inf = infliximab; Met = methotrexate; no BD meds = no systemic BD‐associated medication. Patients on infliximab (n = 1) and methotrexate (n = 1) were excluded in evaluation of effect on NK cells due to only single individuals in each of these subgroups. Numbers in parentheses above each bar indicate number of individuals in each comparison group.