Potentiation of leukotriene B4-mediated inflammatory response by the adenosine antagonist, 8-phenyl theophylline

Abstract

Previous in vitro studies have shown that adenosine (ADO)-induced inhibition of granulocyte function is near maximal at the sub-micromolar concentrations that would be anticipated in normal tissues. If this mechanism is operative in vivo, then antagonizing ADO receptors should potentiate granulocyte-mediated inflammatory responses. To unmask the putative inhibition, the antagonist, 8-phenyl theophylline (8pTHEO), was continuously suffused over the hamster cheek pouch microcirculation, which was observed with intravital bright field and fluorescence microscopy. As an index of inflammation, macromolecular permeability was measured by determining extravasation of fluorescein isothiocyanate-labelled dextran (MW 150,000). In addition, tissue specimens were fixed and stained with hematoxylin and eosin for histological quantification of intravascular and extravascular granulocytes. The tissue was challenged with a 10-40 min topical application of leukotriene B4 (LTB4, 1.1-4nM) or histamine (0.5 or 10 microM) with and without 8pTHEO, at a concentration (8 microM) that attenuated vasodilation evoked by exogenous ADO. These two inflammatory stimuli were chosen because LTB4 evokes a granulocyte-dependent response in the cheek pouch, while histamine evokes a granulocyte-independent response. 8pTHEO potentiated the dose-related increase caused by LTB4 but had no effect on permeability in baseline conditions or on the response evoked by histamine. In baseline conditions, there were fewer than 500 intravascular granulocytes/mm2 microvessel surface area and fewer than 3000 extravascular granulocytes/cm2 tissue surface area. After LTB4 challenge, there was a 3-4 fold increase in the numbers of extra- and intra-vascular granulocytes compared to baseline, but no dose-related relationship could be detected over the concentration range 1.1-4 nM. With 8pTHEO + 1.1 nM LTB4, the granulocyte accumulation was similar to that with LTB4 alone, but there were significantly more intravascular and extravascular granulocytes in the 8pTHEO after 2.5-4 nM LTB4. In context with previous studies, these results suggest that endogenous ADO exerts a tonic inhibitory influence on granulocytes during LTB4 stimulation and this action can be unmasked with methylxanthines.