Influences of interaction between dengue virus type 2-infected human umbilical vein endothelial cells and macrophages on major inflammatory cytokines / 中华微生物学和免疫学杂志

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 periph-eral 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 macro-phages. 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-in-fected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pre-treating 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 de-creased. 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 un-infected group were 1. 46±0. 67 and 1. 60±0. 54, respectively. Expression of IL-6, IL-8, TNF-αand IL-1βat mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-αand 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-infec-ted HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-cul-ture 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-αand IL-1βin 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-αand IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent.