Circulating soluble factor-inhibiting natural killer (NK) activity of fresh peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients

Abstract

This study was performed in order to assess the cytotoxic activity, both natural (NK) and antibody-dependent (ADCC), of PBMC from 38 IBD patients and correlate it with their clinical features. Cytotoxicity assays were performed using sensitive target cells for NK and ADCC activities. In some experiments, highly purified NK cells, obtained both by Percoll density gradient and by co-culturing non-adherent PBMC with RPMI 8866 feeder cells, were used as effector cells. Furthermore, we evaluated NK cell parameters such as number, surface expression of adhesion molecules (CD11a/CD18, CD49d and CD54) and response to different stimuli. We observed a decreased NK cytotoxicity of PBMC from IBD patients, both in ulcerative colitis (UC) and Crohn's disease (CD), independently of the clinical activity of disease. In contrast, the ADCC lytic activity was within normal range. The lower NK cytotoxic activity observed in our IBD patients cannot be related to a decreased number of NK cells, surface expression of adhesion molecules, defective response to IL-2 and maturative defect. Decreased NK activity was induced in PBMC of controls when serum of patients was added and this was unrelated to monocyte-derived modulating factor(s). Our data show a decreased natural killing by fresh PBMC from IBD patients. This lower activity seems to be unrelated to a primary NK cell defect, since purified NK cells exhibited normal levels of killing. It might be hypothesized that serum factors, possibly derived from lymphocytes, with inhibitory properties on NK activity, might be functionally active in the blood of IBD patients, thus modulating NK activity.

Fig. 1

Ability of IL-2 to restore NK activity of PBMC against K562 target cells in IBD patients. The points represent the means of triplicate determinations. The differences between patients and controls (C) before IL-2 stimulation were statistically significant at every considered E:T ratio (*P = 0.007 and *P = 0.004 at E:T ratios 50:1 and 25:1, respectively). No statistically significant differences between the two groups could be observed after stimulation of effector cells with IL-2 (1000 U/ml) for 24 h.

Fig. 2

(a) Cytotoxic activity of highly enriched NK cells from IBD patients and controls (C). Cytotoxic assays were performed by incubating serial dilutions of Percoll-purified NK cells with 5 × 10^{3 51}Cr-labelled K562 target cells. The points represent the means of triplicate determinations. No statistically significant differences were present between patients and controls at any considered E:T ratio. (b) Levels of killing of NK cells from patients (IBD) and C after co-culture with RPMI 8866 feeder cells. NK cells were obtained by co-culturing non-adherent PBMC from buffy coats (4 × 10⁵/ml) with irradiated RPMI 8866 cells (1 × 10⁵/ml) for 10 days. ⁵¹Cr-labelled K562 were used as target cells. The points represent the means of triplicate determinations. In this assay, the lytic activity of NK cells of patients was similar to controls.

Fig. 3

Dose-dependent effect of patient sera (S) on NK activity of PBMC from control subjects (C). Sera of patients were added to cytotoxicity tests performed using PBMC of controls as effector cells and 5 × 10^{3 51}Cr-labelled K562 as target cells. CS20 and CS50: the assay test was performed in the presence of 20% or 50% serum from patients, respectively. The points represent the means of triplicate determinations.