Captopril Combined with Furosemide or Hydrochlorothiazide Affects Macrophage Functions in Mouse Contact Hypersensitivity Response

Figures

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  Figure 1. CHS reaction in mouse recipient of TNP-conjugated macrophages obtained from drug-treated mice. Donors of peritoneal macrophages were treated intraperitoneally with captopril with or without furosemide or hydrochlorothiazide for eight consecutive days. Then, oil-induced macrophages were harvested and conjugated with TNP hapten for 10 min in darkness at room temperature. After washing, TNP-conjugated macrophages were adoptively transferred by intravenous injection into naive recipients. A week later, TNP-macrophage recipients were challenged with a 0.4% PCL to elicit CHS ear swelling, measured with engineer’s micrometer 24 h later. The averaged results of ear-thickness increase, after subtracting the ear thickness increase in non-sensitized but challenged littermate mice, were expressed as delta +/− standard error of the mean. Two-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001, macrophage donors: n = 3 (in each group), macrophage recipients: n = 4 (in each drug-treated mice test group) + negative control n = 3; N = 2.

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  Figure 2. Actively induced CHS reaction in drug-treated mice. Mice were treated intraperitoneally with captopril with or without furosemide or hydrochlorothiazide for eight consecutive days. On the third day, mice were actively sensitized to TNP by topical application of PCL dissolved in a 1:3 mixture of acetone and ethanol on the shaved abdominal skin. Five days later, mice were challenged to elicit CHS ear swelling by topical application of a 0.4% PCL solution in a 1:1 mixture of acetone and olive oil on both sides of both ears. At 2 and 24 h later, the ear swelling was measured with an engineer’s micrometer. The averaged results of ear-thickness increase, after subtracting the ear-thickness increase in non-sensitized but challenged littermate mice, were expressed as delta +/− standard error of the mean. Two-way ANOVA with Tukey’s post hoc test. ** p < 0.01; *** p < 0.005; **** p < 0.001, n = 4 (in each drug-treated mice test group) + negative control n = 3, N = 2.

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  Figure 3. CHS reaction in mouse recipient of PCL-induced CHS effector cells adoptively transferred to drug-treated mice. Recipients of PCL effector cells were treated intraperitoneally with captopril with or without furosemide or hydrochlorothiazide for eight consecutive days, while donors, on the third day, were sensitized with PCL on the shaved abdominal skin. Five days later, lymph node and spleen CHS effector cells were adoptively transferred by intravenous injection into drug-treated recipients. Just after transfer, recipients were challenged with a 0.4% PCL to elicit CHS ear swelling, measured with an engineer’s micrometer 24 h later. The averaged results of ear-thickness increase, after subtracting the ear-thickness increase in non-sensitized but challenged littermate mice, were expressed as delta +/− standard error of the mean. Two-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.005, n = 4 (in each drug-treated mice test group) + negative control n = 3, N = 2.

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  Figure 4. IL-12p40 secretion by macrophages harvested from drug-treated mice. Oil-induced peritoneal macrophages from mice treated with captopril with or without a respective diuretic drug were cultured in standard conditions, in some cases after stimulation with LPS (200 ng). Enzyme-linked immunosorbent assay (ELISA) was used to measure the concentration of IL-12p40 in supernatant collected after 24 h of the culture. Results were expressed as the mean (+/− SD) per group. Two-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001, n = 3, N = 3.

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  Figure 5. Nitric oxide (NO) secretion by macrophages harvested from drug-treated mice. Oil-induced peritoneal macrophages from mice treated with captopril with or without a respective diuretic drug were cultured in standard conditions, in some cases after stimulation with LPS (200 ng). Griess-based colorimetric reaction was then used to measure the concentration of NO/NO2 in supernatant collected after 24 h of the culture. Results were expressed as mean (+/− SD) per group. Two-way ANOVA with Tukey’s post hoc test. **** p < 0.001, n = 3, N = 3.

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  Figure 6. Generation of reactive oxygen intermediates (ROIs) by macrophages harvested from drug-treated mice. Oil-induced peritoneal macrophages from mice treated with captopril with or without a respective diuretic drug were harvested and incubated with luminol or lucigenin, as a chemiluminescent probe, for 15 min at 37 °C. Afterwards, macrophage oxidative burst was stimulated with mouse serum-opsonized zymosan just before the measurement of luminol- or lucigenin-dependent chemiluminescence with a Lucy 1 luminometer, lasting for 75 min. The averaged results of ROI generation were expressed in relative units of luminescence emission (RULE) per second, n = 3, N = 3.