Sphingosine kinase-dependent migration of immature dendritic cells in response to neurotoxic prion protein fragment

Abstract

The concept that circulating dendritic cells mediate neuroinvasion in transmissible spongiform encephalopathies received strong support from recent observations that prion protein is expressed in myeloid dendritic cells. We observed that prion protein fragment 106-126 is a chemoattractant for monocyte-derived immature but not mature dendritic cells. Signaling events in chemotaxis involved enzymes downstream of G(q) protein and were inhibited by blockade of sphingosine kinase, suggesting transactivation of sphingosine-1-phosphate-dependent cell motility by prion protein.

FIG. 1.

Cytofluorometric analysis of DC surface phenotype. A total of 5 × 10⁵ DCs were washed in phosphate-buffered saline-2% fetal calf serum and resuspended in a solution containing 250 μg of human immunoglobulin G per ml, phosphate-buffered saline, and 2% fetal calf serum. After pelleting, DCs were incubated alternatively with 10 μg of anti-CD80 per ml or anti-HLA-DR monoclonal antibodies and the respective isotype-matched control immunoglobulins. After a washing in phosphate-buffered saline-2% fetal calf serum, a 1:40 dilution of fluorescein isothiocyanate-anti-mouse immunoglobulin G in phosphate-buffered saline-2% fetal calf serum was incubated for 30 min at 4°C. Cells were immediately analyzed on a FACScan. Analysis was performed with CellQuest software (BD Biosciences, Mountain View, Calif.).

FIG. 2.

PrP induces chemotaxis in immature DCs and lacks an effect on mature DCs. DCs were allowed to migrate toward various concentrations of PrP_106-126 for 4 h in modified multiwell Boyden chambers with micropore nitrocellulose filters. The mean random migration distances were 40 ± 6.2 μm for immature and 49 ± 8.3 μm for mature DCs, respectively. The chemotaxis index is the ratio between directed and random migration. Statistical analysis was done by the Mann-Whitney U test after Kruskal-Wallis analysis; *, P < 0.05, n = 6. Random migration of mature DCs was inhibited (#, P < 0.05, n = 6). RANTES and 6Ckine were used as positive controls for immature and mature DCs, respectively.

FIG. 3.

DMS inhibits PrP_106-126-induced chemotaxis. DCs were incubated with DMS for 20 min. Chemotaxis toward PrP_106-126 and fMLP was tested after a washing by using modified multiwell Boyden chambers. Mean random migration was 52 ± 7.5 μm. The chemotaxis index is the ratio between directed and random migration. Statistical analysis was done by the Mann-Whitney U test after Kruskal-Wallis analysis; *, P < 0.05, n = 3.