IL-8 SINGLE-CHAIN HOMODIMERS AND HETERODIMERS - INTERACTIONS WITH THECHEMOKINE RECEPTORS CXCR1, CXCR2, AND DARC

Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) hav e been designed to mimic the dimeric form of IL-8 in solution and faci litate the production of heterodimer variants of IL-8. Physical studie s indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-associatio n of the tethered dimers. However, addition of a single disulfide cros slink between the tethered subunits prevented this multimer from formi ng, yielding a species of dimer molecular weight. Crosslinked single-c hain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no pepti de linker. In addition, neutrophil response to these dimers as measure d by chemotaxis or beta-glucuronidase release is similar to that elici ted by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activ ities. Finally, through construction of single-chain heterodimer mutan ts, we show that only the first subunit's ELR motif is functional in t he single-chain variants.