Phenotypical and functional characterization of peripheral blood natural killer cells in spondyloarthropathy patients

Background:Spondyloarthropathies (SpAs) comprise a cluster of inter-related chronic inflammatory rheumatic diseases characterized by axial and peripheral involvement and extra-articular features. The hypotheses underlying the pathogenesis are increasingly questioned and recently a prominent role for natural killer (NK) cells has been advanced. NK cells, a crucial component of the innate immune system, exhibit cytotoxic properties and provide immunoregulatory cytokines, particularly TNF-alpha and IFN-gamma. Their function is regulated by the integration of both activatory and inhibitory signals from a wide range of cell surface receptors. Objective:To focus on phenotypical and functional characterization of peripheral blood (PB) NK cells from SpA patients to better comprehend their pathogenetic role. Methods:We enrolled seven consecutive out-patients with SpAs [six with ankylosing spondylitis fulfilling the modified New York criteria and one with psoriatic arthritis with prominent axial involvement diagnosed by the presence of psoriasis and seronegative arthritis (4 males and 3 females; mean age 46.4 yrs, range 39–68)]. Patients’ PBMCs were isolated from 35 ml heparinized blood by Lymphoprep gradient centrifugation. A comparative analysis of NK and T cell surface antigen expression was performed by staining PB lymphocytes with fluorescence-conjugated monoclonal antibodies directed to MIC A/B, a ligand for an activator receptor, and KIR3DL1, which mediates inhibitory signals, and to subset cell markers such as CD56, CD3, CD4, CD8 and four-color flow cytometric analysis. A sequential gating strategy, based on forward, side scatter and fluorescence parameters was used to evaluate antigen expression on NK and T cells. NK cell cytotoxicity was assessed by using a degranulation assay which measures cell-surface expression of the lisosomal protein CD107a (LAMP-1). PBMCs were incubated with either K562, an NK cell-sensitive erythroleukemia cell line, and with the mouse mastocytoma cell line FcR* (P815) pre-incubated with antibodies directed against NK-cell activating receptors CD16 and NKG2D at an effector to target ratio of 1:1. The degranulation was detected analyzing the expression of CD107a, recently described as a sensitive marker of NK and CD8+T cell degranulation in healthy subjects, using multi-parameter flow cytometry. The intra-cellular production of TNF-alpha and IFN-gamma following 6 hrs PBMCs stimulation with P815 cells pre-incubated with anti-CD16, anti-NKG2D or both antibodies, was quantified using multi-parameter flow cytometry. Analysis was performed using Wilcoxon’s test for matched samples; the significance of any correlation was determined by Spearman’s rank correlation coefficient, where p values less than 0.05 were deemed statistically significant. Results: A relevant increase in the expression of KIR3DL1 on PB NK cells was found in respect to CD4+and CD8+T lymphocytes (p<0.02). Also, stimulation of NK cells with P815 pre-incubated with both anti-CD16 and anti-NKG2D antibodies appeared statistically significant in inducing cytotoxicity in respect to stimulation of P815 pre-incubated with anti-NKG2D only (p<0.014). Similarly, cytotoxicity was significantly appreci-able when P815 were pre-incubated with anti-CD16 only in respect to anti-NKG2D (p<0.014). A positive correlation was found between cytotoxic properties and TNF-alpha production following stimulation of NK cells with P815 pre-incubated with anti-CD16 only (r = 0.8; p<0.04). Conclusions: KIR3DL1 increased expression on NK cells in SpAs, together with the upregulation of CD107a which demonstrates cytotoxic activity, indicate an activated status of NK cells in these patients, supporting a pathogenetic role for this lymphocyte subset. These preliminary results will be integrated with further experiments to better define the functional characterization of NK population in SpAs.