Interleukin-8 (IL-8) is a member of the CXC branch of the chemokine superfamily and activates neutrophils but not monocytes. The related CC chemokine branch, which includes monocyte chemoattractant protein-1 (MCP-1) and RANTES are potent chemoattractants for monocytes but not neutrophils. Examination of the sequences of the CXC chemokines reveals that the highly conserved leucine, corresponding to Leu in IL-8, is always replaced by tyrosine in CC chemokines. There is also a high degree of conservation among the CXC chemokines of the adjacent Val residue, which points out from the same side of the -sheet as Leu. In RANTES, Val is also replaced by a tyrosine. In order to investigate the role of these residues in controlling cell specificity, we have made the single mutants Leu Tyr, Val Tyr and the double mutant Leu Tyr,Val Tyr of IL-8. These proteins have been expressed in Escherichia coli and purified to homogeneity from inclusion body material. All three mutants have lower potency and efficacy in chemotaxis and calcium mobilization assays using neutrophils. The mutants also show lowered affinity to both IL-8 receptors A and B expressed recombinantly in HL-60 cells and to neutrophils in [I]IL-8 competition assays. Additionally, the Leu Tyr mutation introduces a novel monocyte chemoattractant activity into IL-8. We therefore studied the displacement of [I]MIP-1 by IL-8 Leu Tyr from the CC-CKR-1 receptor. The mutant displaces MIP-1 ligand with an affinity only 12-fold less than MIP-1 itself. This suggests that mutations in this region of IL-8 are involved in receptor binding and activation and in the control of specificity between CC and CXC chemokines.

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