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Title: Steroid modulation of neutrophil function
Author: Maclean, Andrew George
ISNI:       0000 0001 3615 6953
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1997
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Dexamethasone, a glucocorticoid, is used extensively in clinical medicine in the treatment of respiratory diseases, notably asthma. This medical effect is probably due in part to a down-regulation of many cytokines, e.g. IL-8. However, the side effects of steroids often may outweigh the beneficial effects, especially if the disease in question is of a chronic nature. This combined with steroid resistance has led to an increase in the use of non-steroidal anti-inflammatory drugs. These tend to be aspirin derivatives, and many have side-effects. I have investigated a factor that is naturally produced by the body which may alleviate the symptoms of inflammation without many of the side- effects, with an aim to providing a viable alternative. A novel steroid induced monocyte derived factor was described twenty years ago by Stevenson as part of an MD thesis submitted to this university. This remains an unusual phenomenon, as most of the effects of steroids are due to a down-regulation of release; here is one of the few examples of the reverse. Although it raised many interesting points about dispersive motility of populations of polymorphonuclear leukocytes (PMN) it did not look at individual cells, nor at many of the interesting properties of neutrophils. One of the known effects of some steroids (though not Dexamethasone), and many NSAIDs, is a reduction of adhesion of PMN to venous endothelium. Human umbilical vein endothelial cells (and bovine aorta endothelium) were used as an ex vivo model for this, and my results show there is a marked decrease in this adhesion. This effect was observed not only on resting endothelium, but also when the endothelial cells had been pre-treated with ILip or thrombin. This decreased adhesion was due to an interaction with the PMN and the factor, as shown by adhesion being reduced when protein coated coverslips were used as the substrate for adhesion. It is suspected that the reasons for this may be due to an inactivation of integrins such as Mac-1, although a role for selectins cannot be ruled out. Recent work by Diaz-Gonzalez suggests that some NSAIDs act by inducing PMN to shed L-selectin. Other effects of this steroid induced monocyte derived factor on human PMN were determined using biological and biochemical techniques. Previous work has shown a novel dispersive effect of this factor on PMN when used in a uniform concentration, and so I decided to look at the moiphology of treated PMN. Using scanning electron microscopy I observed a polarisation of the PMN, but without any raffling of the membrane, a feature that is normally observed with polarisation. This morphology was not observed with control supernatants taken from monocytes cultured in the absence of Dexamethasone. This moiphology was conserved using cells in suspension and adhered to bovine aorta endothelium. The underlying actin cytoskeleton was examined using confocal microscopy, and the microfilamentous airay was noted as being devoid of spikes, observed with activation, for example with fMLP. Late cytoskeletal controlled effects, the release of granule contents, were also investigated, and it was noted that the release of primary granule contents could be inhibited by this factor in a dose dependent manner. This fusion of granules with the plasma membrane is controlled by the activation of numerous tyrosine kinases, and follows a strict order. Secondary granule release was shown to be inhibited also, as assayed by the cleavage of type I collagen, and analysis of SDS gels. The effects on the metabolic burst showed conflicting results with inhibition being present, again in a dose dependent manner, but much of this activity was removed with increasing purification, leaving only very slight inhibition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Respiratory; Asthma; Cytokines