Ischaemic stroke and the recanalization drug tissue plasminogen activator interfere with antibacterial phagocyte function

Abstract

Background: Stroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE. Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here.

Methods: Ex vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy.

Results: MPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release. r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA.

Conclusions: Intracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies. r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated.

Keywords: Hormones; Innate immune response; NETosis; Oxidative burst; Phagocytosis; Stroke; r-tPA.

Fig. 1

NE and MPO in stroke patients versus controls. Stroke patients were analysed and compared to healthy controls. MPO and NE levels were measured intracellularly in granulocytes by flow cytometry (a, c) and are reported as MFI and in the sera of patients using ELISA (b, d) and reported in ng/ml. MPO was estimated on the day of stroke admission (d0), day 1 (d1), day 3 (d3) and day 5 (d5) by flow cytometry (a; n _crtl = 14, n _d0 = 14, n _d1 = 18, n _d3 = 14, n _d5 = 15). The extracellular amount of MPO was measured by ELISA (d0, d1, d3, d5) (b; n _crtl = 11, n _d0 = 8, n _d1 = 12, n_d3 = 18, n _d5 = 17). The intracellular amount of NE was measured on the day after stroke (d1) and on days 3 and 5 (d3, d5) (c, n _crtl = 10, n _d1 = 10, n _d3 = 10, n _d5 = 10). NE in the sera was analysed on the day of stroke admission (d0) and on days 1, 3 and 5 after stroke (d1, d3, d5) (d; n _crtl = 11, n _d0 = 8, n _d1 = 12, n _d3 = 18, n _d5 = 17). *p < 0.05; **p < 0.01; ***p < 0.005. Mean and SD ranges for a and c and medians and interquartile ranges for b and d are given. ANOVA and Bonferroni comparisons as post hoc tests were used for data in a and c; data in b and d were assessed by Kruskal–Wallis test and Dunn’s multiple comparison test

Fig. 2

NET percentage after incubation with adrenaline, noradrenaline, dexamethasone and acetylcholine. Effect of in vitro administration of 1 × 10⁻⁷ M and 1 × 10⁻⁵ M adrenaline (a) and noradrenaline (b); 2.5 × 10⁻⁷ M and 2.5 × 10⁻⁶ M dexamethasone (c); and 5.5 × 10⁻⁶ M and 5.5 × 10⁻⁴ M acetylcholine (d) on the percentage of NET-producing cells. After incubating blood of healthy donors with hormones and acetylcholine for 4 h at room temperature, neutrophils were isolated, and NETs were induced using PMA or fMLP or left unstimulated (n _a,b = 7; n _c,d = 6). *p < 0.05; **p < 0.01; ***p < 0.005. The mean and SD ranges are given. For a–c, ANOVA and Bonferroni comparisons as post hoc tests were used. Data in d were assessed by Friedman test and Dunn’s multiple comparison test. Repeated measures tests were used within each of the three stimulation conditions

Fig. 3

Influence of r-tPA on phagocytosis. PBMCs of healthy donors were incubated with 0.5 or 1 μg/ml r-tPA for 4 h at 37 °C in 5% CO₂. Phagocytosis was then induced using FITC-labelled opsonized E. coli. The percentage of phagocytosing cells (a, b) and the efficacy of phagocytosis defined as the MFI (c, d) were investigated for granulocytes (a, c) and monocytes (b, d) (n = 10). *p < 0.01; ***p < 0.005. The mean and SD ranges are given. Repeated measures ANOVA and Bonferroni comparisons as post hoc tests were used

Fig. 4

Influence of r-tPA on oxidative burst. Oxidative burst analysis was performed using blood from healthy donors for granulocytes (a) and monocytes (b). Blood samples of healthy donors were incubated with 0.5 or 1 μg/ml r-tPA for 4 h at 37 °C in 5% CO₂. The oxidative burst was then induced using fMLP or PMA, and the efficacy (defined as the mean fluorescence intensity, MFI) of ROS was measured by flow cytometry (n = 10). *p < 0.05; **p < 0.01; ***p < 0.005. The mean and SD ranges are given. Repeated measures ANOVA and Bonferroni comparisons as post hoc tests were used within each of the four stimulation conditions