Leishmania major lipophosphoglycan modulates the phenotype and inhibits migration of murine Langerhans cells

Abstract

Langerhans cells (LC), members of the dendritic cell family, play a central role in the initiation and regulation of the immune response against the protozoan parasite Leishmania major. LC take up antigens in the skin and transport them to the regional lymph nodes for presentation to T cells. However, it is not known whether LC functions are modulated by parasite antigens. In the present study, we examined the effect of a major parasite surface molecule, L. major lipophosphoglycan (LPG), on the maturation of LC and their migratory properties. The results show that exposure to LPG did not affect the expression of major histocompatibility complex (MHC) class II and B7, but induced an up-regulation of CD25, CD31 and vascular endothelial (VE)-cadherin expression and a down-regulation of Mac-1 expression, by LC. Importantly, LPG treatment inhibited the migratory activity of LC, as it reduced their efflux from skin explants and their migration in transwell cultures. These results suggest that Leishmania LPG impairs LC migration out of the skin and thus may modulate their immunostimulatory functions, which require LC translocation from skin to lymph nodes.

Figure 1

Flow cytometric analysis of CD25 expression by gated major histocompatibility complex (MHC) class II-positive Langerhans cells (LC) after overnight incubation in the presence (grey lines) or absence (thick black lines) of lipopolysaccharide (LPS), Leishmania major-conditioned medium (LCM) or lipophosphoglycan (LPG). Isotype-matched antibodies were used as controls (thin black lines).

Figure 2

Flow cytometric analysis of major histocompatibility complex (MHC) class II and B7.2 expression by Langerhans cells (LC). Freshly isolated cells or cells cultured overnight in the absence of exogenous mediators or in the presence of lipopolysaccharide (LPS), Leishmania major-conditioned medium (LCM) or lipophosphoglycan (LPG), were stained with anti-I-A^d and anti-B7.2 antibodies and subjected to two-colour fluorescence-activated cell sorter (FACS) analysis.

Figure 3

Flow cytometric analysis of CD31, vascular endothelial (VE)-cadherin and Mac-1 expression by gated major histocompatibility complex (MHC) class II-positive Langerhans cells (LC) after overnight incubation in the presence (grey lines) or absence (thick black lines) of lipopolysaccharide (LPS), Leishmania major-conditioned medium (LCM) or lipophosphoglycan (LPG). Isotype-matched antibodies were used as controls (thin black lines).

Figure 4

Lipophosphoglycan (LPG) reduced the migration of Langerhans cells (LC). (a) Efflux of LC from skin explants cultured in the absence of exogenous mediators or in the presence of lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α) or LPG. Major histocompatibility complex (MHC) class II-positive cells that had migrated from the skin into the medium were detected by fluorescence-activated cell sorter (FACS) analysis (cells in the absence of exogenous mediators versus LPS, P < 0·0001; versus TNF-α, P < 0·002; versus LPG, P < 0·0008). (b) Transwell migration of isolated LC cultured in the absence of exogenous mediators or in the presence of LPS, TNF-α, Leishmania major-conditioned medium (LCM), LPG or phosphoglycan (PG). Cells that had migrated to the bottom chambers were enumerated, and LC were identified by FACS analysis for expression of MHC class II (LC in the absence of exogenous mediators versus LPS, P < 0·0001; versus TNF-α, P < 0·0002; versus LPG, P < 0·001).