The pig as a model for investigating the role of neutrophil serine proteases in human inflammatory lung diseases

Abstract

The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084-34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837-1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.

Figure 1. Characterization of purified porcine blood neutrophils

Flow cytometric analysis of pig (A) and human (B) white blood cells and of purified porcine (C) and human (D) neutrophils. The quiescent porcine neutrophils were >99% pure. SEM (E) of purified quiescent pig and human neutrophils. Porcine white blood cells and quiescent neutrophils were characterized using antibodies raised against the surface marker of leucocytes (SWC1) (F), neutrophils (SWC8) (G) and lymphocytes and monocytes (CD45Ra) (H) (grey peaks), with IgM isotype as control (light grey peaks). gr, granulocyte; ly, lymphocyte; mo, monocyte.

Figure 2. Degranulation of pig neutrophils by the calcium ionophore A23187

SEM and confocal microscopy of quiescent and activated porcine neutrophils (A) showing DNA (blue) and Pr3 (green), chosen here as a representative NSP. Proteolytic activities as measured by the increase in fluorescence units/s of FRET substrates specific for each NSP in suspensions of 1.5×10⁶ /150 μl of pig neutrophils (median±interquartiles, n=10) and human neutrophils (median±interquartiles, n=5) (B). * indicate significant (α=5%) increases over unstimulated cells. FRET substrates were Abz-QPMAVVQSVPQ-EDDnp for NE, Abz-TPFSGQ-EDDnp for cat G and Abz-VADCADYQ-EDDnp for Pr3 [–26].

Figure 3. Inhibition of porcine NSPs by human NSP inhibitors

(A) Percentage inhibition of porcine NSPs in supernatants of A23187-activated neutrophil suspensions (10⁶ cells in 150 μl) by human α1-Pi (10⁻⁷ M final concetration) and human ACT (10⁻⁷ M final concentration) showing that the specificity of human inhibitors for porcine proteases is the same as that for human NSPs (median±interquartiles, n=4). * indicate significant (α=5%) inhibition of NSPs. (B) Irreversible complexes formed between human α1-Pi and porcine Pr3 shown by Western blotting in reducing conditions using anti-human Pr3 antibodies (lanes 3 and 4). Human Pr3 and its complex with α1-Pi were used as a control (lanes 1 and 2). The molecular mass in kDa is indicated on the left-hand side. (C) Inhibition of porcine proteases by reversible inhibitors of human NSPs. The specific inhibition of porcine elastase by EPI-hNE4 (10⁻⁶ M final concentration), the preferential inhibition of NE over Pr3 by P0005259 (10⁻⁶ M final concentration), the inhibition of both NE and cat G by SLPI (10⁻⁶ M final concentration) and the specific inhibition of porcine Pr3 by azapro-3 (5×10⁻⁵ M final concentration) reproduced the results obtained with human NSPs, indicating the functional resemblance between pig and human NSPs (median±interquartiles, n=4). * indicate significant (α=5%) inhibition of NSPs.

Figure 4. Secretion of NETs by porcine blood neutrophils in response to PMA and bacteria

SEM of quiescent porcine neutrophils (A) and of neutrophils activated with PMA (B), S. aureus (C) and P. aeruginosa (D), before (A–D) and after (E–H) DNase treatment. Higher magnification images show the trapping of P. aeruginosa (I) and S. aureus (J) in the fibres of chromatin.

Figure 5. Characterization of NSPs in porcine NET suspensions

Confocal microscopy showing DNA (blue) and Pr3 (green) in suspensions of PMA- and bacterium-triggered neutrophils (A). Arrows denote the co-localization of Pr3 and chromatin of NETs. (B and C) Quantification of DNA and of proteolytic activities in suspensions of PMA-and bacterium-activated cells before (B) and after (C) DNase treatment (median±interquartiles, n=4). * indicate significant (α=5%) decreases in DNA or increases in protease activity induced by DNase.