The Antiseptic and Antineoplastic Agent Taurolidine Modulates Key Leukocyte Functions

Abstract

Background/aim: Although taurolidine is known to exert a wide spectrum of biological actions, its effects on immune cells have not been characterized in detail. In this study, we investigated the ex vivo effects of taurolidine on relevant innate and adaptive immune cell functions.

Materials and methods: Leukocyte functions in whole blood were assessed following treatment with various taurolidine concentrations. Viability of peripheral blood mononuclear cells (PBMCs) and granulocytes was measured using the WST-1 assay. PBMC function was assessed by measuring TNFα and IFNγ production after stimulation with lipopolysaccharide (LPS) or Candida, respectively. Reactive oxygen species (ROS) production by granulocytes was measured in whole blood using luminol-enhanced chemiluminescence. Granulocyte degranulation and activation were evaluated by membrane expression of degranulation (CD63, CD66B) and adhesion markers (CD62L, CD11b) using immunofluorescent staining followed by flow-cytometric analysis.

Results: Taurolidine decreased viability of PBMCs and granulocytes: after 2 h, IC₅₀ concentrations were 500 and 520 μg/ml, respectively. Following prolonged exposure (≥24 h) of PBMCs, the IC₅₀ concentrations declined to 40 μg/ml. PBMC cytokine production significantly decreased at taurolidine concentrations below the cytotoxic threshold, whereas no changes in ROS production were observed. The expression of all granulocyte adhesion and degranulation markers increased at concentrations higher than 500 μg/ml (the cytotoxic level of taurolidine).

Conclusion: Taurolidine exhibits a dose- and time-dependent cytotoxicity toward PBMCs and granulocytes. The effects on PBMCs, as exemplified by a decrease in cytokine production, occurred below the toxic threshold, whereas granulocyte function (ROS production) remained unchanged at these taurolidine concentrations. Granulocyte activation and degranulation markers only increased at cytotoxic taurolidine concentrations.

Keywords: CD11b; CD62L; CD63; CD66B; PBMC; Taurolidine; cell markers; cytokines; cytotoxicity; granulocyte; leukocytes; neutrophil; reactive oxygen species.

Figure 1. Survival of peripheral blood mononuclear cells (PBMCs) and granulocytes after exposure to various taurolidine and/or polyvinylpyrrolidone (PVP) concentrations. Blood samples from one volunteer were exposed to various taurolidine and/or PVP concentrations. Each measurement was performed in triplicate. Dotted lines indicate 50% survival. A, B, C) PBMC survival after 2, 4, 24, and 48 h incubation with two-fold dilution series of A) Taurolidine/PVP in RPMI (10% v/v), B) Taurolidine in RPMI and C) PVP in RPMI. D, E, F) Granulocyte survival after 2 and 4 h incubation in two-fold dilution series of D) Taurolidine/PVP in RPMI (10% v/v), E) Taurolidine in RPMI and F) PVP in RPMI. IC₅₀: Half maximal inhibitory concentration.

Figure 2. Relative cytokine production of peripheral blood mononuclear cells (PBMCs) exposed to taurolidine and/or polyvinylpyrrolidone (PVP). Blood samples from eight volunteers were exposed to various taurolidine and/or PVP concentrations. Each measurement was performed in duplicate. PBMCs were incubated with taurolidine/PVP in RPMI (10% v/v) (A, D, G); taurolidine in RPMI (B, E, H); or PVP/MQ (10% v/v) (C, F, I). PBMCs were stimulated with LPS (A-F) or Candida (G-I), and tumor necrosis factor α (TNFα) (after 4 and 24 h) and interferon-gamma (IFNγ) (after 48 h) were assessed, respectively. Cytokine production for each taurolidine concentration is shown relative to stimulated untreated controls (set at 1). Red bars indicate half maximal inhibitory concentrations (IC₅₀) of taurolidine/PVP or taurolidine, as illustrated in Figure 1. *p<0.05 compared to other taurolidine concentrations or control.

Figure 3. Stimulus-induced reactive oxygen species (ROS) at different taurolidine concentrations in whole blood. Blood samples from twelve volunteers were exposed to taurolidine/polyvinylpyrrolidone (PVP) in RPMI (10% v/v) and stimulated with A) phorbol 12-myristate13-acetate (PMA) or B) serum-treated zymosan (STZ). Each measurement was performed in quintuple. Mean ROS production was expressed as relative light units per granulocyte. The ROS production for each taurolidine/PVP concentration is shown relative to stimulated untreated controls (set at 1). Red bars indicate half maximal inhibitory concentrations (IC₅₀) of taurolidine/PVP or taurolidine, as illustrated in Figure 1. *p<0.05 compared to other taurolidine concentration or control.

Figure 4. Relative expression of cell membrane markers on granulocytes (CD11b, CD62L, CD63, CD66B). Graphs show the mean activation of cell membrane markers from whole blood of eight volunteers exposed to 0, 100, or 500 μg/ml taurolidine/polyvinylpyrrolidone (PVP) in RPMI (10% v/v). The expression of membrane surface antigens in viable granulocytes at each taurolidine concentration is shown relative to stimulated untreated controls (set at 1). A) activation of CD11b, B) activation of CD62L, which becomes downregulated during cell activation due to receptor shedding, C) activation of CD63, and D) activation of CD66B. *p<0.05 compared to other taurolidine/PVP concentration or control.