Conditioned medium from cultured peritoneal macrophages that have phagocytosed a myelin membrane fraction is mitogenic for cultured Schwann cells. Production of the mitogenic supernatant was time-and dose-dependent with a maximal Schwann cell-proliferative response from supernatants after 48-hr incubation of cultured macrophages with myelin-enriched fraction (200 ,ug of protein per ml). The response was specific for myelin membrane: supernatants derived from macrophages incubated with axolemma, liver microsomes, polystyrene beads, or lipopolysaccharide were not mitogenic. Lysosomal processing of the myelin membrane was necessary for the production of the mitogenic factor, which was shown to be heat labile and trypsin sensitive. There was no species specificity because myelin membranes isolated from the central and peripheral nervous systems of rat, bovine, and human were equally potent in eliciting mitogenic supernatant. However, supernatants derived from central nervous system myelin membranes were two to three times more mitogenic than those obtained from peripheral nervous system fractions of the same species. Previous observations that myelin is mitogenic for cultured Schwann cells may, in part, involve the intermediate processing of myelin by macrophages that are present in Schwann cell cultures. These results suggest that macrophages play a crucial role in Schwann cell proliferation during Wallerian degeneration.The source of the mitogenic signal for Schwann cell division during Wallerian degeneration is not clear. Removal of myelin sheaths accompanying Wallerian degeneration has been ascribed to Schwann cells (1, 2) as well as macrophages (3-5). Beuche and Friede (5) observed that Wallerian degeneration proceeding without nonresident phagocytic cells (macrophages) showed no Schwann cell proliferation and no active intracellular digestion of myelin-implying that myelin membranes were removed solely by macrophages after nerve degeneration. This observation also suggests that Schwann cell proliferation is related to myelin removal by macrophages. Salzer and Bunge (6) using dorsal root ganglion explant cultures showed that only myelin-related Schwann cells proliferate after axotomy. In contrast, however, in vivo studies by Pellegrino et al. (7) (9). The presence of macrophages in our Schwann cell cultures and the possible involvement of macrophages in removal of myelin debris during Wallerian degeneration (10) prompted us to study the role of macrophages in mediating mitogenicity of myelin for cultured Schwann cells. Using rat peritoneal macrophages, we have shown that macrophages stimulated with MEF produce a soluble factor(s) mitogenic for cultured Schwann cells. Production of the soluble mitogenic factor shows a time-and dose-dependent response. Other membrane fractions or nonspecific agents do not stimulate macrophages to produce a mitogenic-conditioned medium. The myelin membrane undergoes lysosomal processing in the macrophage before mitogenic factor is produced. Sensitivity of the mitogenic sup...

Tetanus toxin is known to bind neuronal tissue selectively. To study the interactions of this potent neurotoxin in an intact cell system, the binding of 125I-tetanus toxin was characterized in a neuroblastoma retina hybrid cell line, N18-RE-105. The binding of 125I-tetanus toxin to membranes prepared from N18-RE-105 cells showed many similarities to the interactions of 125I-toxin with rat synaptic membranes. The binding was decreased with increasing temperature, ionic strength, and pH. 125I-Toxin bound to membranes with high affinity: KD = 0.62 +/- 0.05 nM; Bmax = 196 +/- 45 pmol/mg protein. Quantitative thin-layer chromatography and acid-degradation analysis revealed that N18-RE-105 cells contained polysialogangliosides GD1a and GT1b in high concentrations. An assay was developed to quantitate surface-bound and internalized 125I-tetanus toxin by exploiting the observation that surface-bound 125I-toxin is susceptible to pronase digestion. When cells were incubated with 125I-tetanus toxin at 0 degree C, all of the bound 125I-toxin could be degraded with pronase. In contrast, when the incubations were performed at 37 degrees C, within 10 min about 50% of the total cell-associated 125I-toxin was pronase-resistant. Temperature pulse experiments demonstrated that 125I-tetanus toxin that was bound to cells at 0 degree C rapidly disappeared from the surface when the cells were warmed to 37 degrees C, as revealed by the appearance of pronase-resistant radioactivity. This internalization was sensitive to metabolic inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

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The release of membrane-associated growth factors after neural injury may influence the outcome of the recovery. For example, for remyelination to occur after neural injury it is critical for the glial cell to proliferate prior to remyelination in both the PNS and CNS. In the CNS, the relative response of the oligodendrocytes and astroglia to growth factors mobilized during neural injury may play a role in the cellular dynamics of repair of neural injury or scarring and subsequent failure to repair neural injury. In support of this view, we have studied the mitotic potential and cell cycle kinetics of cultured adult oligodendrocytes and found that these adult cells respond only weakly to factors such as FGF which are known to be potent mitogens for neonatal cells. However, given the same dose of FGF, adult astrocytes are mitotically stimulated to a much greater degree than are the adult oligodendrocytes (Vick and De Vries, unpublished observations). Given the pathways which may be operative in the release of growth factors after injury, it has not escaped our attention that, provided the released factors are in equilibrium with easily accessible and peripheral body fluids, these released factors may serve as new markers for neural injury. Further experiments are in progress to explore this possibility.