Neutrophils contain prolyl endopeptidase and generate the chemotactic peptide, PGP, from collagen

Abstract

Prolyl endopeptidase (PE), a protease that cleaves after proline residues in oligopeptides, is highly active in brain and degrades neuropeptides in vitro. We have recently demonstrated that PE, in concert with MMP's, can generate PGP (proline-glycine-proline), a novel, neutrophil chemoattractant, from collagen. In this study, we demonstrate that human peripheral blood neutrophils contain PE, which is constitutively active, and can generate PGP de novo from collagen after activation with LPS. This novel, pro-inflammatory role for PE raises the possibility of a self-sustaining pathway of neutrophilic inflammation and may provide biomarkers and therapeutic targets for diseases caused by chronic, neutrophilic inflammation.

Figure 1

Indicated amounts of human neutrophil lysate (10⁶ cells) were assayed for PE enzymatic activity (expressed as mU/ml). Results are presented as mean ± SEM (A) and compared using the two group t test. Increased PE activity was observed with greater amounts of neutrophil lysate (* p < 0.05 compared with 0μl lysate, n = 3 per group). Results are representative of several experiments. Cytospins of human peripheral blood neutrophils on glass slides were incubated with anti-PE antibody (B), anti-PE antibody pre-adsorbed with PE (C) or pre-immune rabbit antibody (D), followed by FITC-labeled goat anti-rabbit IgG, and examined by immunofluorescence microscopy. PE in human neutrophils was located in the cytoplasm in a granular pattern (B).

Figure 2

Human neutrophil lysate was separated by SDS-PAGE, transferred to nitrocellulose membranes and probed for PE (A). Lanes 1, 2 and 3 are a coomassie blue stained gel of MW markers, human PMN lysate (18 μg protein per lane) and rhPE respectively. Lanes 4, 5 and 6 are a chemiluminescence developed Western using our rabbit anti-PE antibody corresponding to Lanes 1, 2 and 3. PE in human neutrophils was a monomer and migrated at 75kDa, similar to rhPE. PCR for PE was performed on total RNA isolated from human peripheral blood neutrophils and the product run on a 2% agarose gel (B). Human PE primers should amplify a 95bp product. PCR for actin was performed concurrently as a positive control. Lanes: Invitrogen 1Kb+ ladder (1), negative control (2), 95bp PCR product from human PMN (3 and 4), 95bp PCR product from pTrcHisB vector containing rhPE (5), negative control (6), 200bp PCR product for actin (7 and 8).

Figure 3

Previously dialyzed Type 1 collagen was incubated with human peripheral blood neutrophils (1 × 10⁶) with and without LPS (100μg/ml) for 1.5 hours. Supernatants were passed through a 10kDa filter and analyzed by ESI-LC-MS/MS for PGP and N-α-PGP. Levels of PGP and N-α-PGP generated are presented as mean ± SEM and compared using the two group t test. LPS-stimulated neutrophils generated significantly greater amounts of PGP and N-α-PGP (* p < 0.01 compared with collagen alone or collagen incubated with neutrophils without LPS, n = 6 per group).