Phenotype and functions of natural killer cells in critically-ill septic patients

Abstract

Rationale: Natural killer cells, as a major source of interferon-γ, contribute to the amplification of the inflammatory response as well as to mortality during severe sepsis in animal models.

Objective: We studied the phenotype and functions of circulating NK cells in critically-ill septic patients.

Methods: Blood samples were taken <48 hours after admission from 42 ICU patients with severe sepsis (n = 15) or septic shock (n = 14) (Sepsis group), non-septic SIRS (n = 13) (SIRS group), as well as 21 healthy controls. The immuno-phenotype and functions of NK cells were studied by flow cytometry.

Results: The absolute number of peripheral blood CD3-CD56(+) NK cells was similarly reduced in all groups of ICU patients, but with a normal percentage of NK cells. When NK cell cytotoxicity was evaluated with degranulation assays (CD107 expression), no difference was observed between Sepsis patients and healthy controls. Under antibody-dependent cell cytotoxicity (ADCC) conditions, SIRS patients exhibited increased CD107 surface expression on NK cells (62.9[61.3-70]%) compared to healthy controls (43.5[32.1-53.1]%) or Sepsis patients (49.2[37.3-62.9]%) (p = 0.002). Compared to healthy (10.2[6.3-13.1]%), reduced interferon-γ production by NK cells (K562 stimulation) was observed in Sepsis group (6.2[2.2-9.9]%, p<0.01), and especially in patients with septic shock. Conversely, SIRS patients exhibited increased interferon-γ production (42.9[30.1-54.7]%) compared to Sepsis patients (18.4[11.7-35.7]%, p<0.01) or healthy controls (26.8[19.3-44.9]%, p = 0.09) in ADCC condition.

Conclusions: Extensive monitoring of the NK-cell phenotype and function in critically-ill septic patients revealed early decreased NK-cell function with impaired interferon-γ production. These results may aid future NK-based immuno-interventions.

Trial registration: NTC00699868.

Trial registration: ClinicalTrials.gov NCT00699868.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Evaluation of cytotoxic functions of NK cells in ICU patients.

Correlation between the direct cytotoxicity CFSE-based assay and the degranulation CD107a expression assay to evaluate cytotoxic functions of NK cells in ICU patients (n = 14). Results are expressed as % lysis of target cell for the CFSE-assay, and as % NK-cell expressing CD107a for the degranulation assay. Effector–target ratio is 50/1 (PBMC/K562) for the CFSE-assay, and 2.5/1 (NK/K562) for the CD107a expression assay.

Figure 2. Evaluation of NK cell functions in ICU septic patients.

NK degranulation (A) and intracellular production of IFN-γ (B) of ICU patients with Sepsis, SIRS, and healthy controls. A: Degranulation responses by CD107a cell-surface expression (% of positive NK cells) against K562 target cells (natural cytotoxicity) or P815 mouse mastocytoma cells coated with rabbit anti-mouse lymphocyte antibodies (ADCC). B: Intracellular IFN-γ expression (percentage of positive NK cells), against K562 target cells or P815 (ADCC). Number of samples from each group: Sepsis group (n = 29), SIRS group (n = 13), and healthy controls (n = 21). A black bar inside the box-and-whiskers plots indicates the median. p(kw): Comparison between healthy, SIRS and Sepsis groups by Kruskal-Wallis test. p: pairwise comparisons between groups (healthy, SIRS, Sepsis) by Kruskal-Wallis post–hoc methods for multiple comparisons adjusted by step-up Simes method.