A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation

Abstract

Chronic neutrophilic inflammation is a manifestation of a variety of lung diseases including cystic fibrosis (CF). There is increasing evidence that fragments of extracellular matrix proteins, such as collagen and elastin, play an important role in inflammatory cell recruitment to the lung in animal models of airway inflammation. Unfortunately, the association of these peptides with human disease and the identification of therapeutic targets directed toward these inflammatory pathways have remained elusive. In this study, we demonstrate that a novel extracellular matrix-derived neutrophil chemoattractant, proline-glycine-proline (PGP), acts through CXC receptors 1 and 2 on neutrophils, similar to N-acetylated proline-glycine-proline (N-alpha-PGP). We describe the specific multistep proteolytic pathway involved in PGP generation from collagen, involving matrix metalloproteases 8 and 9 and prolyl endopeptidase, a serine protease for which we identify a novel role in inflammation. PGP generation correlates closely with airway neutrophil counts after administration of proteases in vivo. Using CF as a model, we show that CF sputum has elevated levels of PGP peptides and that PGP levels decline during the course of CF inpatient therapy for acute pulmonary exacerbation, pointing to its role as a novel biomarker for this disease. Finally, we demonstrate that CF secretions are capable of generating PGP from collagen ex vivo and that this generation is significantly attenuated by the use of inhibitors directed toward matrix metalloprotease 8, matrix metalloprotease 9, or prolyl endopeptidase. These experiments highlight unique protease interactions with structural proteins regulating innate immunity and support a role for these peptides as novel biomarkers and therapeutic targets for chronic, neutrophilic lung diseases.

FIGURE 1

a, PGP acted via a CXCR-dependent mechanism to cause neutrophil chemotaxis: PMN are pretreated with CXCR1 and CXCR2 Abs or IgG2a isotype control Ab (2 μg/ml) for 1 h at 22°C. PGP (at 10 μg/ml) is placed in the bottom of chemotaxis plate. The isotype Ab demonstrated no change in neutrophil chemotaxis compared with untreated cells (■). However, at 1 μg/ml concentration of each CXCR Ab, PGP chemotaxis is completely blocked (*, p < 0.01 compared with no Ab and isotype Ab control). b, N-α-PGP is increased in CF samples compared with normal control samples: CF (n = 10) and normal control (n = 10) sputum samples were analyzed using ESI-LC/MS/MS for N-α-PGP detection. CF samples demonstrated 8 (80%) of 10 positive for N-α-PGP vs normal controls having 1(10%) of 10 positive for N-α-PGP. The threshold for positivity (0.825 ng/ml) was determined as two SDs above mean (95% confidence interval) for control sputum values.

FIGURE 2

a, PE activity was elevated in CF samples compared with normal controls: CF (n = 10) and normal control (n = 10) sputum samples were examined for PE activity using a colorimetric assay. CF samples demonstrated a 5-fold increase in PE activity compared with normal controls (*, p < 0.01). b, PE activity correlated with PGP: concentration of PGP was correlated with PE activity in CF samples (n = 8). The samples demonstrated a correlation coefficient (R²) of 0.718 (p < 0.01).

FIGURE 3

a, PGP production was significantly increased in CF samples compared with normal control samples on type I collagen: CF sputum (n = 10) and normal control sputum (n = 10) were each incubated on extensively dialyzed type I collagen for 24 h at 37°C. PGP values of the samples on PBS were subtracted from PGP values of samples incubated on type I collagen to determine PGP production. PGP generated from CF samples were significantly increased compared with normal control samples on type I collagen (*, p < 0.05). b, PGP production was significantly increased in CF samples compared with normal control samples on type II collagen: CF sputum (n = 10) and normal control sputum (n = 10) were each incubated on extensively dialyzed type II collagen for 24 h at 37°C. PGP values of the samples on PBS were subtracted from PGP values of samples incubated on type II collagen to determine PGP production. PGP generated from CF samples were significantly increased compared with normal control samples on type II collagen (*, p < 0.05).

FIGURE 4

PE, MMP-8, and MMP-9 inhibitors can block the production of PGP: inhibitors were incubated for 6 h with pooled CF sputum and these sputa were placed on type I collagen for 24 h as previously described. PGP concentrations from these groups were compared with pooled CF sputum on type I collagen not treated with inhibitor. PE inhibitor demonstrated complete blockade of PGP production and MMP-8- and -9-specific inhibitors individually demonstrated 80–90% inhibition, with their combination resulting in complete blockade of PGP generation. Doxycycline, a nonspecific MMP inhibitor, demonstrated comparable PGP inhibition as MMP-8 alone. Neither MMP-2- nor HNE-specific inhibitors resulted in significant changes in PGP production.

FIGURE 5

a, In vivo PGP generation: in vivo PGP production was examined using MMPs or HNE with or without PE. Various proteases (50 μl) were administered intratracheally into murine (4- to 6-wk-old BALB/c mice) airways and bronchoalveolar lavage fluid was collected 24 h later. PGP levels were determined using ESI LC-MS/MS. PGP production was significantly increased in MMP-9 with PE (*, p < 0.05 vs PBS control; †, p < 0.05 vs MMP-9 alone) and MMP-8 with PE (*, p <0.05 vs PBS control; †, p <0.05 vs MMP-8 alone) compared with other proteases with or without PE. PBS control and PE alone had similar PGP production. Of note, aminophenylmercuric acetate alone (or in combination with PE) also did not generate PGP (data not shown). Number of mice per group = 6. b, PGP production correlated with neutrophil influx: PGP production levels (■) were compared with PMN counts ( ) in mice treated with a combination of the indicated protease and PE from Fig. 3a. There is a notable correlation between PGP production and PMN counts for each condition (R² = 0.996, inset).

FIGURE 6

PGP levels decline during inpatient therapy for CF exacerbation: sputum PGP levels from CF individuals (n = 12) were examined within 48 h of admission and at discharge (day 13/14) for CF exacerbation. The mean levels of PGP decreased during hospitalization (146.4 ± 24.4 vs 80.0 ± 22.5; *, p < 0.01), although these levels are still 5-fold elevated compared with secretions from normal controls (†, p < 0.01).

FIGURE 7

PGP generation is a multistep process: the generation of PGP is a multistep process initially involving release of MMP-8 or MMP-9 from activated neutrophils. These proteases denature and proteolytically cleave collagen to fragments 30 –100 aa in length. These collagen fragments are then further cleaved to PGP by PE. The PGP generated then acts as a neutrophil chemoattractant and allows for an environment of ongoing proteolytic damage and PGP generation.