Leukocyte adhesion has been implicated as the critical event leading to organ dysfunction during inflammation. However, the experimental support for the linkage between leukocyte adhesion and increased microvessel permeability has been inconsistent. Our preliminary studies suggest that leukocyte adhesion as single cells does not result in increased microvessel permeability. Instead, we found that leukocyte-platelet aggregation and aggregate adhesion produced a prolonged permeability increase. The overall aim of this project is to clarify the interaction between leukocyte recruitment and permeability increase. We propose to identify the initiating steps and critical factors contributing to increased permeability during acute inflammation using a newly developed method that combines single microvessel perfusion with autologous blood perfusion. This approach allows the mechanisms that regulate microvessel permeability to be studied when endothelium interacts directly with blood cell elements, and also retains the capability of single vessel perfusion for precise measurements of vascular permeability under well-controlled experimental conditions. The proposed research is to test two hypotheses. 1) The formation of platelet/leukocyte aggregates and agents released from the aggregates are critical for leukocyte-dependent increases in microvessel permeability and the activated platelets play a central role, and 2) the increased microvessel permeability induced by either leukocyte/platelet/endothelial cell interactions or inflammatory mediators occurs through a mechanism that involves decreased cellular cAMP levels. Leukocyte adhesion and migration and leukocyte/platelet/endothelium interactions will be induced by systemic and local application of cytokines and/or inflammatory mediators with autologous blood perfusion. Changes in permeability will be determined by paired measurements of hydraulic conductivity, or solute permeability coefficient before and after leukocyte or leukocyte/platelet aggregate adhesion in the same microvessel. To correlate functional studies with morphological changes, a combination of in vivo silver staining, immunofluorescence staining with confocal microscopy, and electron microscopy will be used to examine the junction changes between endothelial cells, the location of adherent and transmigrating leukocytes and the corresponding spatial distribution of adhesion molecules in microvessel walls under the same experimental conditions whereby permeability is studied. Our newly developed experimental approach overcomes certain limitations of previous in vivo methods and will advance the knowledge in the field.
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