Functional Response of Neutrophils in the Presence of LPS: Aspects of Locomotion and Metabolism in Normal and Diabetic Cells

Immunology

Noah, Rahim Md

University:University of Glasgow

关键词: Immunology;   

Others  :  http://theses.gla.ac.uk/78032/1/11003385.pdf

来源: University of Glasgow

【 摘 要 】

This thesis reports a study of effects of bacterial lipopolysaccharides (LPS) on morphological polarization, locomotion and chemiluminescence of human neutrophi. leukocytes (polymorphonuclear leukocytes:PMN). Change from a spherical to a polarized morphology by a cell is the initial event in cell locomotion. PMN in suspension were shown to change shape even in the presence of small amounts of LPS (≥100 ng/ml) in the medium. Exposing PMN to LPS from rough strains of bacteria resulted in slow shape change (60-90 minutes), unlike chemotactic factors which cause shape change within a few minutes. This suggests an indirect effect. Rough strains were more active than the smooth chemotypes in inducing polarization of PMN. The number of polarized cells increased as the period of incubation increased implying the possible presence of a second (non-LPS) agonist that promoted further polarizing activity. Polymyxin B sulfate (10mug/ml) was added to the cells prior to challenging with LPS (10mug/ml) in order to inhibit this biological effect. However, polymyxin in this system did not reduce the number of polarized PMN to a statistically significant extent. To test if a chemotactic-type factor might have been released by the cells, supernatant was prepared from PMN (10e6 cells/ml) which had been incubated with LPS for 90 minutes. The supernatant (but not LPS alone) was shown to cause the shape change within 10 minutes in a shortterm polarization assay on a new batch of PMN. By reducing the cell density in the presence of constant amounts of LPS (10 mug/ml), it could be shown that polarizing activity of the supernatant was reduced. To assess the relationship of shape change to that of locomotory ability, three additional leukocyte locomotion assays were employed. With a micropore filter assay, migration of the cells in different concentration gradients of the factor showed that the released material behaved like a chemotactic factor as analyzed by the checkerboard system. A collagen gel assay was used to study detailed cell locomotion quantitatively (population of cells migrating) and also qualitatively (morphological changes during locomotion). Detailed behavioural analysis of the PMN response to LPS was obtained from visual studies. Quantitative measurements of shape change showed a slowly developing increase in elongation of the cells exposed to LPS for up to 90 minutes, in contrast to rapid elongation of cells exposed to the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). One of the ways a neutrophil might release a stimulant is through arachidonic acid metabolism. Steroidal and non-steroidal antiinflammatory drugs used to modulate the shape change process of the leukocytes displayed varied effects. Two drugs, with reported inhibitory effects on lipoxygenase activity, BW755C and Revlon 5901A reduced the number of PMN polarized by LPS. Conversely, neither indomethacin nor dexamethasone had any effect. Preliminary characterization of the cell released factor was also conducted. The factor was heat stable and non-dialyzable. Hexane extraction, thin layer chromatography and column gel filtration suggested that the released material was heterogenous. A low molecular weight factor (about 2000 kD) was identified by gel filtration on Sephadex G-25. Monoclonal antibodies against the CD11/CD18 family of cell surface receptors (shown by other researchers to be responsible for the binding of LPS to PMN) were used as inhibitors of LPS binding to PMN. Some antibodies were relatively effective in inhibiting LPS-induced PMN polarization particularly those against CD11b/CD18. Combinations of monoclonals against three antigens (CD11a, b and c) were more effective than any one alone. The study using these antibodies unexpectedly showed that the binding site of LPS to the phagocyte is highly affected by antibodies to the alpha-chain and not the beta-chain as reported by previous investigators. Competitive inhibitory binding of LPS to PMN was observed with synthetic peptides containing an RGD-sequence. The observation points to a probable involvement of the sequence in LPS-induced neutrophil polarization. Chemiluminescence was used an additional test of LPS activation of PMN. On addition of LPS (10 mug/ml) , slow but progressive light emission was noted. Normal peaking time ranged from 30 to 60 minutes with different intensities from different donors. Addition of platelet activating factor (PAF) as a primer did not enhance the response but instead slightly depressed the activity. LPS on its own can also act as a priming agent .