Anti-Inflammatory Activities of Captopril and Diuretics on Macrophage Activity in Mouse Humoral Immune Response

Abstract

Hypertension is accompanied by the over-activation of macrophages. Diuretics administered alone or in combination with hypotensive drugs may have immunomodulatory effects. Thus, the influence of tested drugs on mouse macrophage-mediated humoral immunity was investigated. Mice were treated intraperitoneally with captopril (5 mg/kg) with or without hydrochlorothiazide (10 mg/kg) or furosemide (5 mg/kg) by 8 days. Mineral oil-induced peritoneal macrophages were harvested to assess the generation of cytokines in ELISA, and the expression of surface markers was analyzed cytometrically. Macrophages were also pulsed with sheep red blood cells (SRBC) and transferred to naive mice for evaluation of their ability to induce a humoral immune response. Tested drugs increase the expression of surface markers important for the antigen phagocytosis and presentation. SRBC-pulsed macrophages from mice treated with captopril combined with diuretics increased the secretion of antigen-specific antibodies by recipient B cells, while macrophages of mice treated with hydrochlorothiazide or furosemide with captopril increased the number of antigen-specific B cells. Tested drugs alter the macrophage secretory profile in favor of anti-inflammatory cytokines. Our results showed that diuretics with or without captopril modulate the humoral response by affecting the function of macrophages, which has significant translational potential in assessing the safety of antihypertensive therapy.

Keywords: antihypertensive therapy; diuretics; humoral immunity; hypotensive drugs; macrophages.

Scheme 1

General scheme of experiments. Mice were administered with captopril with or without furosemide or hydrochlorothiazide for 8 days. On day 3, peritoneal exudate was induced with mineral oil. Peritoneal macrophages were harvested 5 days later and either cultured to assess cytokine secretion, analyzed cytometrically for surface marker’s expression, or pulsed with sheep red blood cells (SRBC) and transferred into naive mice, from which blood sera and spleens were collected a week later to measure the titer of SRBC-specific antibodies or to enumerate splenic SRBC-specific B cells in the plaque-forming assay (PFA), respectively.

Figure 1

Captopril and diuretic drugs impact humoral immune response in mice. Donors of macrophages were treated intraperitoneally with captopril with or without furosemide or hydrochlorothiazide for eight days. Then, oil-induced peritoneal macrophages, after pulsing with sheep red blood cells (SRBC), were transferred intraperitoneally into recipients, from which sera and spleens were collected individually seven days later. (a) Number of plaque-forming cells (PFC) expressing SRBC-specific antibody-producing B cells in spleens of macrophage recipients was estimated through a plaque-forming assay and indicated as a mean number of PFC per spleen. (b) SRBC-specific antibody titers in sera of recipient mice were assessed through a direct hemagglutination assay and expressed as a mean value of log2 of antibody titer. Two-way or three-way ANOVA with Tukey post hoc test. * p < 0.05; ** p < 0.01; ns—not significant.

Figure 2

Captopril and diuretic drugs impact the expression of macrophage surface markers. (a) The level of expression of markers of phagocytosis (CD14, CD11b, CD16/32), and (b) antigen presentation, including I-A^k (MHC class II), CD80, CD86, and CD40, was cytometrically assessed on the surface of oil-induced peritoneal macrophages from mice treated for eight days with captopril and/or respective diuretic drug. Results were shown as a mean percentage (with SD) of macrophages expressing a particular marker within either the total population of analyzed macrophages or their Mac3+ subpopulation. Two-way ANOVA with Tukey post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.005.

Figure 2

Captopril and diuretic drugs impact the expression of macrophage surface markers. (a) The level of expression of markers of phagocytosis (CD14, CD11b, CD16/32), and (b) antigen presentation, including I-A^k (MHC class II), CD80, CD86, and CD40, was cytometrically assessed on the surface of oil-induced peritoneal macrophages from mice treated for eight days with captopril and/or respective diuretic drug. Results were shown as a mean percentage (with SD) of macrophages expressing a particular marker within either the total population of analyzed macrophages or their Mac3+ subpopulation. Two-way ANOVA with Tukey post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.005.

Figure 3

Captopril and diuretic drugs influence the secretion of cytokines by cultured macrophages. Oil-induced peritoneal macrophages from mice treated with captopril with or without respective diuretic drugs were cultured in standard conditions, in some cases after stimulation with LPS (200 ng). Enzyme-linked immunosorbent assay (ELISA) was used to measure (a) the concentration of IL-6, and TNFα in supernatants collected after 24 h of culture and (b) the concentration of IL-10 and TGF-β1 in supernatants collected after 48 h of culture. Concentrations were expressed as mean (+/− SD) per group. Two-way ANOVA with Tukey post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.

Figure 3

Captopril and diuretic drugs influence the secretion of cytokines by cultured macrophages. Oil-induced peritoneal macrophages from mice treated with captopril with or without respective diuretic drugs were cultured in standard conditions, in some cases after stimulation with LPS (200 ng). Enzyme-linked immunosorbent assay (ELISA) was used to measure (a) the concentration of IL-6, and TNFα in supernatants collected after 24 h of culture and (b) the concentration of IL-10 and TGF-β1 in supernatants collected after 48 h of culture. Concentrations were expressed as mean (+/− SD) per group. Two-way ANOVA with Tukey post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.