Macrophage inflammatory protein-1alpha (MIP-1alpha) 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-1alpha is temporally expressed with neutrophils at sites of
infection, we examined proteolysis of
MIP-1alpha in vitro by the neutrophil-derived
serine proteases:
cathepsin G,
elastase, and
proteinase 3. Recombinant human
MIP-1alpha isoforms LD78beta and LD78alpha 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 LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. 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 Phe24-Ile25 and Tyr28-Phe29, whereas
elastase and
proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these
proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from
Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated
serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in
Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.