Inhibition of DC-SIGN-mediated trans infection of T cells by mannose-binding lectin

Abstract

Some dendritic cells (DC) express a cell-surface lectin called 'dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin' (DC-SIGN). DC-SIGN has been shown to mediate a type of infection called 'trans' infection, where DC bind human immunodeficiency virus (HIV) and efficiently transfer the virus to T cells. We investigated the possibility that mannose-binding lectin (MBL), a soluble lectin that functions as a recognition molecule in innate immunity and that binds to HIV, could block trans infection mediated by DC-SIGN. Binding studies with glycoprotein (gp)120/gp41-positive and -negative virus preparations suggested that DC-SIGN and MBL bind primarily to glycans on gp120/gp41, as opposed to glycans on host-cell-derived proteins, indicating a close overlap in the binding site of the two lectins and supporting the notion that MBL could prevent binding of HIV to DC-SIGN. Preincubation of X4, R5 or dual-tropic HIV strains with MBL prevented DC-SIGN-mediated trans infection of T cells. The mechanism of MBL blocking trans infection of T cells was at least partly caused by blocking of virus binding to DC-SIGN positive cells. This study shows that MBL prevents DC-SIGN-mediated trans infection of T cells in vitro and suggests that in infected persons, MBL may inhibit DC-SIGN-mediated uptake and spread of HIV.

Figure 1

Mannose-binding lectin (MBL) structure and dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin (DC-SIGN) expression by THP cells. (a) Purified recombinant MBL and serum MBL were analysed by Western blotting with anti-MBL monoclonal antibody (mAb) after electrophoresis on 8% polyacrylamide gels under non-reducing conditions. (b) Expression of DC-SIGN by DC-SIGN-positive (THPD) and -negative (THP) THP cells was determined by flow cytometric analysis of cells stained with either DC28 anti-DC-SIGN mAb (Ab) or an isotype-matched control mAb (C) followed by incubation with fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG.

Figure 2

Binding of human immunodeficiency virus (HIV) to dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) is dependent on glycoprotein (gp)120/gp41 glycans. Equal amounts (5000 pg of p24) of either gp120/gp41-positive (Env+) or gp120/gp41-negative (Env–) virus were incubated with THP cells expressing DC-SIGN (DC-SIGN) or in MBL-coated microtitre wells (MBL). After incubation, unbound virus was removed by washing, and bound virus detected by p24 enzyme-linked immunosorbent assay (ELISA) after lysis in detergent. Data shown represent mean values ± standard deviation (SD) from one experiment representative of three different experiments.

Figure 3

Mannose-binding lectin (MBL) inhibits human immunodeficiency virus (HIV) trans infection and binding mediated by dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin (DC-SIGN). HIV_GP (a) or HIV_BAL (b) were incubated with THP cells expressing DC-SIGN (THPD) or DC-SIGN-negative THP cells (THP) either in the absence of inhibitors or in the presence of mannan (THPD + Man) or the presence of MBL. Unbound virus was removed by washing and THP cells were then co-cultured with phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs). The level of p24 in culture supernatants was measured on day 10. (c) and (d) THP cells were incubated with virus as in (a) and (b) and the amount of p24 antigen associated with THPD and THP cells was determined by enzyme-linked immunosorbent assay (ELISA) after lysis in detergent. Data shown represent mean values ± standard deviation (SD) from one experiment representative of three different experiments (for HIV_GP) or two different experiments (for HIV_BAL).

Figure 4

Comparison of mannose-binding lectin (MBL) with other compounds for inhibition of human immunodeficiency virus (HIV) binding to dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin (DC-SIGN)-positive cells. THP cells expressing DC-SIGN were incubated with virus in the presence of MBL, bovine serum albumin, transferrin, phytohaemagglutinin (PHA) or Cyanovirin-N, and the amount of p24 antigen associated with cells was determined. Data shown represent mean values from one experiment representative of three different experiments.