Gangliosides of myelosupportive stroma cells are transferred to myeloid progenitors and are required for their survival and proliferation

In previous studies, we have shown that the myelopoiesis dependent upon myelosupportive stroma required production of growth factors and heparan-sulphate proteoglycans, as well as generation of a negatively charged sialidase-sensitive intercellular environment between the stroma and the myeloid progenitors. In the present study, we have investigated the production, distribution and role of gangliosides in an experimental model of in vitro myelopoiesis dependent upon AFT-024 murine liver-derived stroma. We used the FDC-P1 cell line, which is dependent upon GM-CSF (granulocyte/macrophage colony-stimulating factor) for both survival and proliferation, as a reporter system to monitor bioavailability and local activity of GM-CSF. GM3 was the major ganglioside produced by stroma, but not by myeloid cells, and it was required for optimal stroma myelosupportive function. It was released into the supernatant and selectively incorporated into the myeloid progenitor cells, where it segregated into rafts in which it co-localized with the GM-CSF-receptor α chain. This ganglioside was also metabolized further by myeloid cells into gangliosides of the a and b series, similar to endogenous GM3. In these cells, GM1 was the major ganglioside and it was segregated at the interface by stroma and myeloid cells, partially co-localizing with the GM-CSF-receptor α chain. We conclude that myelosupportive stroma cells produce and secrete the required growth factors, the cofactors such as heparan sulphate proteoglycans, and also supply gangliosides that are transferred from stroma to target cells, generating on the latter ones specific membrane domains with molecular complexes that include growth factor receptors.

Mucosal Adjuvant Properties of Mutant LT-IIa and LT-IIb Enterotoxins That Exhibit Altered Ganglioside-Binding Activities

ABSTRACT LT-IIa and LT-IIb, the type II heat-labile enterotoxins of Escherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and E. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

Non-plasmalemmal localisation of the major ganglioside in sea urchin eggs

SummaryM5 ganglioside (NeuGcα2–6Glcβl-' Cer) is the predominant glycosphingolipid in sea urchin eggs. Distribution of M5 ganglioside was studied in unfertilised and fertilised eggs of the sea urchin Hemicentrotus pulcherrimus by indirect immunofluorescence microscopy. In the cortices of unfertilised eggs, anti-M5 antibody strongly stained the submembranous, polygonal and tubular network of endoplasmic reticulum that was revealed by a membrane-staining dye, DiIC18(3). In addition to the cortical network of endoplasmic reticulum, at least two morphologically distinct vesicles were positive to the antibody. In the cortices isolated from fertilised eggs 30 min after insemination, the antibody stained only a similar network of endoplasmic reticulum, presumably the one reconstructed 5–10 min after fertilisation. During mitosis the endoplasmic reticulum is known to aggregate within the asters of the mitotic apparatus. Indeed, the antibody stained the asters and (more strongly) the vesicular components attaching to the periphery of the mitotic apparatus.

Disialoganglioside GD2 distributes preferentially into substrate-associated microprocesses on human melanoma cells during their attachment to fibronectin.

Human melanoma cells (M21) actively attach and spread on a fibronectin substrate. Indirect immunofluorescence assays with specific monoclonal antibodies directed to the disialoganglioside GD2, the major ganglioside expressed on M21 melanoma cells, indicate that during the cell attachment process this molecule redistributes into microprocesses that make direct contact with the fibronectin substrate. Scanning and transmission immunoelectron microscopic studies with anti-GD2 monoclonal antibodies and immuno-gold staining demonstrate that GD2 preferentially localizes into substrate-associated microprocesses that emanate from the plasma membrane of the M21 cells. Staining with monoclonal antibodies directed to other melanoma surface antigens fails to demonstrate a similar distribution pattern on these cells. Direct evidence is provided that GD2 is involved in M21 cell attachment to fibronectin, since treatment of these cells with anti-GD2 monoclonal antibodies causes cell rounding and detachment from a fibronectin substrate. Moreover, scanning electron microscopy demonstrates that this loss of attachment of fibronectin is characterized by a perturbation of the cell attachment-promoting microprocesses that in the presence of these antibodies lose contact with the fibronectin substrate.

Differences in liver ganglioside patterns in various inbred strains of mice

The ganglioside patterns in the liver of different inbred and hybrid strains of mice were investigated. The inbred strains were Balb/cAnNCr1BR, C57BL/6NCr1BR, DBA/2NCr1BR. C3H/HeNCr1BR; the hybrid strain was the Swiss albino. The following major gangliosides were found to be present in mouse liver: GM3-NeuAc; GM3-NeuGl, GM2 [a mixture of one species carrying N-acetylneuraminic acid (NeuAc) and one carrying N-glycollylneuraminic acid (NeuGl)], GM1 and GD1a-(NeuAc, NeuGl). The qualitative and quantitative patterns of liver gangliosides were markedly different in the various inbred strains of mice; in Balb/cAnNCr1BR strain, ganglioside GM2 was preponderant (99.2% of total ganglioside content); in C57BL/6NCr1BR, the major ganglioside was GM2 (90.4%), followed by GM3-NeuAc (5.6%) and GM3-NeuGl (4.0%); in DBA/2NCr1BR, GM2 accounted for 77.1%, GD1a-(NeuAc, NeuGl) 18.9% and GM1 3.1% of gangliosides; in C3H/HeNCr1BR, GM2 constituted 50.6%, GM1 22.8% and GD1a-(NeuAc, NeuGl) 22.1%. In the hybrid Swiss albino mice, liver ganglioside composition markedly varied from one animal to another, GM3-NeuGl, GM2 and GD1a-(NeuAc, NeuGl) being the predominant gangliosides in the various cases.