Proteolysis of Macrophage Inflammatory Protein-1α Isoforms LD78β and LD78α by Neutrophil-derived Serine Proteases

Macrophage inflammatory protein-1α (MIP-1α) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1α is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1α in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1α isoforms LD78β and LD78α were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78β and LD78α were cleaved by neutrophil lysates at Thr 16 -Ser 17 , Phe 24 -Ile 25 , Tyr 28 -Phe 29 , and Thr 31 -Ser 32 . This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe 24 -Ile 25 and Tyr 28 -Phe 29 , whereas elastase and proteinase 3 cleaved at Thr 16 -Ser 17 and Thr 31 -Ser 32 . Proteolysis of LD78β resulted in loss of chemotactic activity. The role of these proteases in LD78β and LD78α degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefèvre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78β and LD78α. These findings suggest that severe periodontal tissue destruction in Papillon-Lefèvre syndrome may be related to excess accumulation of LD78β and LD78α and dysregulation of the microbial-induced inflammatory response in the periodontium.