Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines

Abstract

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells (HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor (M-CSF) to prepare macrophages. The purity of CD14+ CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-infected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pretreating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2.66±0.53, P<0.05) and then decreased. The purity of macrophages detected by flow cytometry was (89.16±2.07) %. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16.10±0.17 and 29.76±0.58, while the expression levels at 24 h in the uninfected group were 1.46±0.67 and 1.60±0.54, respectively. Expression of IL-6, IL-8, TNF-α and IL-1β at mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-α and IL-1β expression at 24 h were 45.82±3.72, 52.34±1.69 (12 h), 8.94±1.75 and 30.96±1.44 in the infected macrophages, and 1.16±0.22, 1.15±0.21, 1.11±0.09 and 1.47±0.31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infected HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-culture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0.01) and that of IL-6, IL-8, TNF-α and IL-1β in macrophages (P<0.01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-α and IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent. Key words: Dengue virus type 2 (DENV-2); Human umbilical vein endothelial cell (HUVEC); Macrophage; Cytokine; Immunoregulation