Activation of B Cell Locomotion In Vitro

Komai-Koma, Mousa (1995) Activation of B Cell Locomotion In Vitro. PhD thesis, University of Glasgow.

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This project looks at the signals that induce locomotion in resting B cell populations and in germinal centre B cells, both from human tonsil. Signals that induce locomotion in blood B cells compared with high-density tonsil B cells were also studied. Polarization studies of the response of cells from immunized mice to antigen are also included. B cells were purified from tonsil by established procedures to yield a high- density fraction (resting cells out of cycle) and low-density (activated) fraction. Germinal centre cells were present in, and were purified from, the latter fraction. Cells from these fractions were assayed for locomotor activity. Two methods were used to study the locomotor activity of B cells; (1) Polarization assay. Measurement of shape-change from a spherical to a polarized shape on stimulation with an attractant. (2) Invasion of collagen gels. Lymphocytes overlaid on a collagen gel containing an attractant will migrate into the gel in larger numbers than into control gels. Previous studies of T cells showed that the full development of the capacity for locomotion and chemotaxis in lymphocytes requires two stages, (a) Resting cells require to be cultured with a growth activator and move from G0 into the G1 phase of cell cycle. After overnight culture, a locomotor population of cells is obtained, (b) These cells are now capable of responses to chemoattiactants and show immediate (<30 min) polarization and locomotion when incubated in their presence. (1) High density B cells. These are small surface IgM+ and surface IgD+ cells which are not in cycle. When freshly purified from the tonsil, very few of these cells show locomotor capacities. The results presented here demonstrate that culture overnight in IL-4, aCD40 or lL-13 induces locomotor shape change in a high proportion of high-density B cells. The proportion of polarized cells increases slowly over a period of 24-48 hours suggesting that locomotor capacity is activated as the cells pass from G0 to the G1 phase of growth. Anti-IL-4 and anti-IL-13 inhibit the locomotion induced by their respective cytokines. IFN-gamma inhibits the locomotion response induced by IL-4. Culture in combination of IL-4 and aCD40 stimulates polarization of more cells than culture in either alone. A combination of IL-4, aCD40, and aIgM stimulates polarization of still more cells (up to 60-70% of the population). Adding aIgM to cultures with aCD40, or with IL-4 does not increase the polarization significantly compared with either aCD40 or IL-4 alone. In addition to study of the effect of locomotor activators on locomotion in overnight culture, the immediate (<30min.) effects of attractants on locomotion of the resting B cell fraction were studied, using either cells direct from the tonsil or cultured B cells. The polyclonal activators, algD and aIgM, were tested in short term assays (30 min. incubation) on freshly isolated cells and on cells cultured in IL-4. Both populations showed immediate polarization to anti-Ig, but cultured cells responded more strongly than cells direct from the tonsil. The optimum attractant concentration of anti-Ig was 100ng-1mug/ml. There was no response to the appropriate isotype controls, mouse IgG2a and sheep Ig. Cells cultured in IL-4 also polarized in a short-term assay to aCD40 (100ng-1mug/ml) within 30 minutes but not to isotype control mouse IgG 1. The results suggest that in contrast to IL-4 and IL-13, anti-CD40 acts not only as a locomotor activator but also as a chemoattractant. Cells direct from the tonsil showed no chemoattractant response to anti-CD40. To measure locomotion itself, cells cultured in IL-4 were layered on top of collagen gels incorporating aIgM, aIgD, aCD40, and HBSS alone, and were allowed to invade for 18 hours. The number of cells invading gels incorporating any of the three stimuli was greater than that invading gels containing medium alone. FACS analysis on invaded cells show that 75 +/- 12% of cells were IgM+ and 82% IgD+. The gel invasion assay selects the locomotor population and demonstrates clearly that small resting IgM+ and IgD+ B cells not only change- shape in response to anti-IgM and anti-IgD, but also show invasive locomotion in response to these antibodies. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Peter Wilkinson
Keywords: Immunology
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-76315
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 15:46
Last Modified: 19 Nov 2019 15:46

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