Effects of low molecular weight heparin on the polarization and cytokine profile of macrophages and T helper cells in vitro

Abstract

Low molecular weight heparin (LMWH) is widely used in recurrent miscarriage treatment. The anti-coagulant effects are established, while immunological effects are not fully known. Our aim was to assess LMWH effects on activation and polarization of central regulatory immune cells from healthy women, and on placenta tissues from women undergoing elective abortions. Isolated blood monocytes and T helper (Th) cells under different activation and polarizing conditions were cultured with or without LMWH. Flow cytometry showed that LMWH exposure induced increased expression of HLA-DR and CD206 in macrophages. This phenotype was associated with increased secretion of Th17-associated CCL20, and decreased secretion of CCL2 (M2-associated) and CCL22 (Th2), as measured by multiplex bead array. In accordance, LMWH exposure to Th cells reduced the proportion of CD25highFoxp3+ regulatory T-cells, intensified IFN-γ secretion and showed a tendency to increase the lymphoblast proportions. Collectively, a mainly pro-inflammatory effect was noted on two essential tolerance-promoting cells. Although the biological significancies of these in vitro findings are uncertain and need to be confirmed in vivo, they suggest the possibility that immunological effects of LMWH may be beneficial mainly at an earlier gestational age to provide an appropriate implantation process in women with recurrent miscarriage.

Figure 1

Effects of LMWH on macrophage polarization. Effect of LMWH on macrophage phenotype markers, measured as proportion of cells (%) and relative expression of the entire population (median fluorescence intensity, MFI). Macrophages were cultured for 6 days with GM-CSF or M-CSF in the absence or presence of 1 or 10 IU LMWH. Data are expressed as mean ± S.D. One way ANOVA and Sidak’ s post hoc test: *p < 0.05; **p < 0.005; n = 8.

Figure 2

Effects of LMWH on the production of chemokines by macrophages. Macrophages were cultured with GM-CSF and M-CSF for 6 days in the absence or presence of 1 or 10 IU LMWH. Data are expressed as box plots showing median and inter-quartile range (box) and range (whiskers). When Friedman test was p < 0.05, Wilcoxon test was performed: *p < 0.05; n = 7.

Figure 3

Effects of LMWH on Th cells. Effects of LMWH on Th cell phenotype (A), cytokine secretion (B) and viability and activation (C,D), measured as proportion (%) of viable cells and of lymphoblasts. CD4⁺ T cells, both unstimulated and stimulated with anti-CD3 and anti-CD28 Abs, were cultured for 3 days in the absence or presence of 1 or 10 IU LMWH. Data are expressed as mean ± S.D. (A,C,D) and as box plots (B) showing interquartile range (box) and range (whiskers). One way ANOVA and Sidak’ s test: *p < 0.05 (A). Friedman and Wilcoxon tests (B): *p < 0.05. One way ANOVA and Sidak’ s test (C,D); (A) n = 7, (B) n = 19 (un-exposed) and n = 17 (exposed to LMWH), (C) n = 18 (un-exposed and exposed to 1 IU LMWH in T cell stimulated samples), n = 13 (exposed to 1 IU LMWH in T cell unstimulated samples), n = 10 (exposed to 10 IU LMWH in T cell unstimulated samples), n = 15 (exposed to 10 IU LMWH in T cell stimulated samples), (D) n = 15.

Figure 4

Effects of LMWH on 1^st trimester placental explants. Effects of LMWH on the production of soluble factors by placental explants. Placenta explants were exposed or not to 1 or 10 IU of LMWH for 20–24 hours. Data are expressed as median and inter-quartile range (box) and range (whiskers). Friedman test followed by Wilcoxon; n = 10.