Lack of galectin-3 speeds Wallerian degeneration by altering TLR and pro-inflammatory cytokine expressions in injured sciatic nerve.

Wallerian degeneration (WD) comprises a series of events that includes activation of non-neuronal cells and recruitment of immune cells, creating an inflammatory milieu that leads to extensive nerve fragmentation and subsequent clearance of the myelin debris, both of which are necessary prerequisites for effective nerve regeneration. Previously, we documented accelerated axon regeneration in animals lacking galectin-3 (Gal-3), a molecule associated with myelin clearance. To clarify the mechanisms underlying this enhanced regeneration, we focus here on the early steps of WD following sciatic nerve crush in Gal-3(-/-) mice. Using an in vivo model of nerve degeneration, we observed that removal of myelin debris is more efficient in Gal-3(-/-) than in wild-type (WT) mice; we next used an in vitro phagocytosis assay to document that the phagocytic potential of macrophages and Schwann cells was enhanced in the Gal-3(-/-) mice. Moreover, both RNA and protein levels for the pro-inflammatory cytokines IL-1ß and TNF-α, as well as for Toll-like receptor (TLR)-2 and -4, show robust increases in injured nerves from Gal-3(-/-) mice compared to those from WT mice. Collectively, these data indicate that the lack of Gal-3 results in an augmented inflammatory profile that involves the TLR-cytokine pathway, and increases the phagocytic capacity of Schwann cells and macrophages, which ultimately contributes to speeding the course of WD.

Efficient uptake of mannosylated proteins by a human Schwann cell line.

Complex carbohydrate structures are essential molecules of infectious microbes and host cells, and are involved in cell signaling associated with inflammatory and immune responses. The uptake of mannose-tailed glycans is usually carried out by macrophages, dendritic cells (DCs), and other professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation, and to promote T cell effector responses. Since Schwann cells (SCs) have been proposed as immunocompetent cells, we investigated whether a human cell line (ST88-14 cells) could bind mannosylated ligands in a specific manner. The saturation of uptake of mannosylated molecules by ST88-14 cells and the internalization and distribution pathway of these ligands were tested by cytometry and confocal plus electron microscopy, respectively. This uptake showed a dose-dependent increase, the saturation point being reached at high concentrations of mannosyl residues/240 mM mannose. Merging of man/BSA-FITC and S100 labeling showed their partial, but, significant colocalization. Ultrastructural analysis of ST88-14 cells after incubation with HRP-colloidal gold, without or with subsequent chasing at 37C, showed an initial location on the cell surface and temperature- and time-dependent internalization of the probe. Our findings suggest an efficient mannosylated ligand uptake system through putative lectin(s) that may be operational in inflammatory and immune responses.

Mycobacterium leprae infection of human Schwann cells depends on selective host kinases and pathogen-modulated endocytic pathways.

Mycobacterium leprae, an obligate intracellular pathogen, shows a unique tropism for Schwann cells (SC). This leads to the peripheral neuropathy disorder observed in leprosy. In this study, we investigated signal transduction events and the intracellular fate of M. leprae during the interaction of the microorganism with SC. First, we demonstrated that the human schwannoma cell line ST88-14 readily phagocytized the bacteria as observed by time-lapse microscopy, actin staining and electron microscopy. The effect of specific kinase inhibitors on M. leprae internalization was then investigated showing that functional protein tyrosine kinase, calcium-dependent protein kinase and phosphatidylinositol 3-kinase, but not cAMP-dependent protein kinase are essential for phagocytosis of the bacteria. Similar results were obtained when irradiated and live bacteria were compared and when M. leprae was pre-coated with recombinant histone-like-protein/laminin binding protein, a bacterial adhesin. In addition, experiments were performed to analyze the bacterial trafficking within the endosomal network by labeling the acidified intracellular compartments of M. leprae-infected SC with the Lysotracker acidotrophic probe. Acidification of vesicles containing live M. leprae was minimal in both RAW murine macrophages and SC, although phagosomes containing heat-killed bacteria seem to follow normal endocytic maturation. These data indicate that the invading bacteria interfere with normal endocytic pathway maturation of bacteria-containing phagosomes within SC.