Some aspects of IL-8 pathophysiology. III: Chemokine interaction with endothelial cells.

Chemokines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilized on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin-8 (IL-8) and RANTES, but not monocyte inflammatory protein-1 alpha on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL-8 binding patterns. The subcellular distribution of IL-8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL-8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL-8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL-8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL-8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL-8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel-Palade bodies.

Production of neutrophil activating peptide-1 (NAP-1/IL-8) by blood and synovial fluid mononuclear cells from patients with arthritis.

As polymorphonuclear leukocytes (PMN) are predominant in inflammatory synovial fluids, we investigated the production of neutrophil-activating peptide-1 (NAP-1) by mononuclear cells (MC) from 15 synovial fluids and matched peripheral blood. MC were cultured for 24 h alone or with stimulants (ConA, LPS). NAP-1 was determined in the supernatants by a bioassay (elastase release from normal human PMN) and an immunoassay (sandwich ELISA with a mouse anti-NAP-1 mAb and an alkaline phosphatase labelled goat anti-NAP-1 pAb). The results showed a significant increase in NAP-1 production by synovial fluid MC when compared to peripheral blood MC. Both cell types produced more NAP-1 in the presence of added stimuli. The results obtained with the two methods of detection were in close agreement. No relationship was found between the amount of NAP-1 produced in 24 h and the number of synovial fluid leukocytes, the erythrocyte sedimentation rate, the diagnosis of the underlying arthritis or the treatment of the patients.

Immunofluorescence assay for the quantitative and qualitative evaluation of intracellular interleukin-8 in microtiter plates.

In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular protein (colorimetric assay). The quantitative and qualitative determination of intracellular IL-8 was achieved by immunofluorescence using the ELF-detection system (Molecular Probes, Eugene, OR), which is approximately 10 times more sensitive than conventional immunofluorescence detection systems. Thus, a 32x objective magnification (without immersion oil) is sufficient to precisely assess the subcellular localization of IL-8. Experiments were carried out with human umbilical vein endothelial cells. Drugs interfering with transcription or translation and inhibitors of protein kinase C inhibited both production and secretion of IL-8. Brefeldin A (BFA), colchicine, and the HMGCoA-reductase inhibitor fluvastatin disrupted the characteristic Golgi localization of IL-8, but only BFA inhibited its secretion. This assay can therefore be used to distinguish drugs that inhibit both IL-8 synthesis and secretion from those that inhibit IL-8 secretion only. It can easily be adapted to other cellular proteins for which a sensitive detection method is required.