Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils.

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called ser-IL-872) and the other 77 amino acids (an N-terminal extended form herein called ala-IL-877). IL-8 derived from T lymphocytes and monocytes is predominantly ser-IL-872, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in ala-IL-877. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert ala-IL-877 to ser-IL-872. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave ala-IL-877, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125Iala-IL-877 neutrophils exhibited a twofold preference for ser-IL-872 over ala-IL-877. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, ser-IL-872 was approximately 10-fold more potent than ala-IL-877 in these assays (ED50 approximately 0.3 nM for ser-IL-872 vs approximately 3 nM for ala-IL-877. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [ser-IL-872 (ED50 greater than 10 nM) was two- to three-fold more potent than ala-IL-877], although in this regard they were less active than FMLP. Our data suggest that ala-IL-877 and ser-IL-872 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the ser-IL-872 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.