CCR5 expression and beta-chemokine production during placental neonatal monocyte differentiation

Abstract

The stage of maturation of monocytes affects their susceptibility to HIV infection. The beta-chemokines and their receptor CCR5 play a crucial role in inflammatory reactions and HIV infection. We therefore examined the correlation between the expression of CCR5 and beta-chemokine production and the susceptibility to HIV infection during cord monocyte (CM) differentiation into macrophages. CM and CM-derived macrophages (CMDM) were examined for beta-chemokine and CCR5 expression. The susceptibility of the CM cultured in vitro at different time points to HIV infection was also determined. Although the levels of CCR5 mRNA expression in freshly isolated CM are comparable to those in CMDM, CM had significantly lower levels of CCR5 protein on the cell surface than CMDM did. Steady increase of CCR5 protein expression on the cell surface was observed during CM differentiation into macrophages. The CCR5 expression correlated with the increased susceptibility to HIV infection by CMDM. Although there was no significant difference in endogenous beta-chemokine production between CM and CMDM, HIV infection of CMDM significantly enhanced production of macrophage inflammatory protein-1alpha and -1beta. CCR5 receptor plays a critical role in HIV infection of neonatal blood monocyte/macrophages.

Figure 1

(A) Flow cytometry analysis of CCR5 surface protein expression on the membrane of freshly isolated CM and CMDM. CM (d 0) and CMDM (d 8) were stained with a mouse MAb against CCR5 (2D7). The shaded histogram represents control staining with isotype-matched antibody (IgG 2a). The open histogram represents CCR5 expression using the 2D7. The results are shown as the percentage of CCR5-positive cells and are representative of five cord blood samples. (B) Effect of stage of differentiation of CM on CCR5 surface protein expression. The cells were harvested at the indicated time above and stained with a monoclonal mouse antibody to CCR5 (2D7) or an isotype-matched antibody (IgG 1) for flow cytometry. The results shown are mean ± SD of duplicate cultures and are representative of five experiments using the cells from five different cord blood samples.

Figure 2

RT-PCR analysis of CCR5 mRNA during differentiation of cord monocytes to macrophages. Cord monocytes were prepared and cultured as described in “Materials and Methods.” Total cellular mRNA was extracted from the cultured cells at the indicated time and subjected to RT-PCR assay using specific primers for (A) CCR5 and (B) β-actin. Representative results from five different cord blood samples are shown.

Figure 3

Production of MIP-1α and MIP-1β in CM and CMDM. Freshly isolated (d 0) CM and 8-d-cultured CMDM were treated with LPS (100 ng/mL) and culture supernatants were collected 24-h post LPS treatment. The results shown are mean ± SD of triplicate cultures and are representative of five experiments using the cells from five different cord blood samples.

Figure 4

Production of MIP-1α and MIP-1β in HIV-infected CM and CMDM. Freshly isolated (d 0) CM and 8-d-cultured CMDM were infected with HIV Bal stain. Culture supernatants were collected for MIP-1α and MIP-1β analysis 4 d postinfection. Results are representative of four experiments using the cells from four different cord blood samples.

Figure 5

HIV infection of placental CM and CMDM. Freshly isolated cord monocytes (d 0 CM) and 8-d-cultured cord monocyte-derived macrophages (d 8 CMDM) were infected with HIV Bal strain. Culture supernatants were collected at the indicated time points after HIV infection (d 4, 8, 12), and assayed for HIV RT activity. Each graph represents an individual cord blood sample (cord 1, 2, 3, and 4).