THE CHEMOKINES IL-8, MONOCYTE CHEMOATTRACTANT PROTEIN-1, AND I-309 ARE MONOMERS AT PHYSIOLOGICALLY RELEVANT CONCENTRATIONS

The chemokines are a family of immune mediators involved in a wide ran ge of inflammatory processes, most importantly as chemoattractants of monocytes, neutrophils, lymphocytes, and fibroblasts to sites of infla mmation. Nuclear magnetic resonance and x-ray crystallographic studies have shown that IL-8 and macrophage-inflammatory protein-1 beta (MIP- 1 beta) form noncovalent dimers and that platelet factor-4 (PF-4) form s noncovalent dimers and tetramers, leading to the assumption that, as a family, the chemokines would form multimeric structures. In this st udy, we analyze the association states of the chemokines IL-8, monocyt e chemoattractant protein-1 (MCP-1), and I-309, by using a combination of size exclusion HPLC, sedimentation equilibrium ultracentrifugation , and chemical cross-linking. We find that the association states of M CP-1 and IL-8 are characterized by an equilibrium between monomers and dimers: although dimers predominate at concentrations above 100 mu M, these chemokines are almost exclusively monomeric at the nanomolar co ncentrations at which they display maximal chemotactic activity. I-309 , by contrast, remains a monomer at all concentrations tested. 1-309 c ontains two additional cysteine residues (C26 and C68) that are not fo und in any other members of the chemokine family. We used cyanogen bro mide and trypsin digestion strategies to demonstrate that these two re sidues are linked in a unique intramolecular disulfide bond. Furthermo re, by using site-directed mutagenesis, we show that the integrity of this bond is crucial for protein secretion.