Blackford, Jeanette; (1996) A study of the rabbit leucocyte integrins and their role in cell adhesion. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

It is clear that the leucocyte integrins play an important role in many cell-cell interactions within the immune system. While such interactions may be necessary for an effective immune response, they must be tightly controlled since the inappropriate activation of leucocytes, as in autoimmune diseases, results in pathology. An understanding of the molecular interactions and activation states of the leucocyte integrins is required to allow development and evaluation of therapeutic strategies. The rabbit provides a suitable species in which to study these processes. A new rabbit CD8+ T cell line, BJ/873, has been characterised by flow cytometry and together with the rabbit CD4+ T cell line, RL-5, has been used to produce a panel of mAb which recognise a variety of rabbit cell surface antigens. mAb which recognise rabbit CD 18, CD 11a and CD 11c have been produced and characterised using cell-surface immunofluorescence and flow cytometry, immunohistochemical staining of rabbit lymphoid tissues. Western blotting and immunostaining, metabolic and cell-surface labelling of cell lines followed by immunoprecipitation and N-terminal amino-acid sequence analysis of immunoaffinity purified proteins. Using these, and previously characterised mAb, the expression of the leucocyte integrins by rabbit lymphoid tissues was investigated by flow cytometry. Following stimulation with phorbol-ester the RL-5 cells were found to homotypically aggregate and inhibition studies revealed that this aggregation is LFA-1 dependent. A number of the anti-LFA-1 mAb produced also stimulated LFA-1 dependent aggregation of the RL-5 cells. The ligand for rabbit LFA-1 in this system was not identified. Three anti-CD11c mAb induced homotypic aggregation of BJ/873 cells. mAb blocking studies showed that anti-CD 18 and anti-ICAM-1 mAb were inhibitory and suggested that in the rabbit, pl50,95 acts as a ligand for ICAM-1. The BJ/873 cells co-express LFA-1 and pi50, 95 as well as other adhesion molecules. Before a definitive statement concerning the interaction of rabbit pi50, 95 with ICAM-1 can be made, these molecules must be expressed in isolation. A probe for rabbit CD 18 was produced by PCR and used to screen a rabbit spleen cDNA library. A single clone containing a 2.3kb insert was selected and sequenced. The clone does not code for the N-terminal portion of CD 18 and this region has been amplified by PCR and sequenced.

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