Locomotion of defined lymphocyte subsets in relation to inflammation

Newman, Ian (1993) Locomotion of defined lymphocyte subsets in relation to inflammation. PhD thesis, University of Glasgow.

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Abstract

This project examined whether expression of defined CD45R isoforms was related to the capacity of human blood lymphocytes to show locomotion in vitro. In addition the role of IL-2 and IL-8 in stimulating such locomotion was investigated. Established assays of lymphocyte locomotion were utilised, i.e. examination for morphological polarization, assessment of invasion into collagen gels, and measurement of cell size in relation to locomotion. A range of stimulators of locomotion was used, and locomotion of lymphocytes expressing CD45RO or CD45RA was assessed. The locomotion of lymphocytes defined by other surface markers, such as CD29 (the VLA-4 B chain), CD3, CD4, CD8, CD19, CD22 and CD25 was also examined in conjunction with expression of CD45R isoforms. Adaptation of methods. The following methods were developed during the project: (1) adaptation of a method which enables separation of lymphocytes on the basis of invasion of collagen gels, such that a range of surface markers on the invasive and non-invasive populations could be detected using FACS analysis; (2) the development of a method enabling the expression of surface markers to be detected by modified APAAP staining, in 21 conjunction with the polarization assay; and (3) the adaptation of both of these methods to enable the size of lymphocytes within defined subsets to be determined using forward scatter values derived from FACS analysis, or cell area measurement in conjunction with the polarization assay. Results. In polarization assays a significantly greater proportion of CD45RO+ (and CD45RA-), than of CD45RA+ (and CD45RO-), lymphocytes became polarized in response to all stimuli used. The stimuli used were HESS, FCS, colchicine, IL-2 and IL-8 for lymphocytes on day 0; HBSS, IL-2 and IL-8 for lymphocytes cultured overnight in FCS; and culture of mononuclear cells for 72 hours in HBSS/HSA, -CD3, PPD, Con A, allo-MLR, PHA or PWM, in which case lymphocytes respond in situ to factors released into the culture supernatant. In collagen gel invasion assays, a consistently greater proportion of CD45RO+ (and of CD29+) lymphocytes, than of CD45RA+ cells invaded collagen gels incorporating their culture supernatants, after culture of mononuclear cells in PPD or allo-MLR. This was the case for both CD4+ and CD8+ lymphocytes, whether invasion proceeded for 3.5 hours or overnight. However no such differential locomotion of lymphocytes expressing different CD45R isoforms was evident if mononuclear cells were cultured in -CD3 or Con A. After culture in -CD3, but not in PPD, marginally more CD4+ than CD8+ lymphocytes were found to invade collagen gels incorporating their culture supernatants. Experiments comparing the response of T and B lymphocytes in collagen gel invasion assays (and in polarization assays) were restricted since it was observed that culture of mononuclear cells in -CD3 led to down-regulation of surface CD3. Recombinant human IL-8 was found to stimulate locomotion into collagen gels of lymphocytes cultured in -CD3. Antibodies to IL-8 blocked the ability of supernatants from mononuclear cells cultured in ?-CD3 to stimulate lymphocyte locomotion into collagen gels. These results suggest a pivotal role for IL-8 in the activity of such supernatants.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Peter Wilkinson
Keywords: Immunology
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-72859
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72859

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