Papillon-Lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses
Ole E. Sørensen, … , Finn Cilius Nielsen, Niels Borregaard
Ole E. Sørensen, … , Finn Cilius Nielsen, Niels Borregaard
Published September 17, 2014
Citation Information: J Clin Invest. 2014;124(10):4539-4548. https://doi.org/10.1172/JCI76009.
View: Text | PDF
Research Article

Papillon-Lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses

Abstract

Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease–deficient immune cell populations, PLS patients do not exhibit marked immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally localize to azurophil granules, including the major serine proteases, elastase, cathepsin G, and proteinase 3, were absent. Accordingly, neutrophils from this patient were incapable of producing neutrophil extracellular traps (NETs) in response to ROS and were unable to process endogenous cathelicidin hCAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent in mature neutrophils, indicating that CTSC mutation promotes protease degradation in more mature hematopoietic subsets, but does not affect protease production in progenitor cells. Together, these data indicate CTSC protects serine proteases from degradation in mature immune cells and suggest that neutrophil serine proteases are dispensable for human immunoprotection.

Authors

Ole E. Sørensen, Stine N. Clemmensen, Sara L. Dahl, Ole Østergaard, Niels H. Heegaard, Andreas Glenthøj, Finn Cilius Nielsen, Niels Borregaard

×

Figure 2

Functional studies on PLS neutrophils.

Options: View larger image (or click on image) Download as PowerPoint
Functional studies on PLS neutrophils. (A) Flow cytometry of nonstimulat...
(A) Flow cytometry of nonstimulated and fMLF-stimulated (10–8 M) neutrophils from PLS and a normal control demonstrating equal disappearance of CD62L and upregulation of CD11b in response to stimulation. Respiratory burst was quantified as DHR-positive cells by flow cytometry of isolated neutrophils stimulated by fMLF (10–8 M). (B) NETosis. Neutrophils were stimulated with PMA or glucose oxidase (GO) to undergo NETosis and stained with DAPI and antibodies against MPO. NETosis was quantified as an increase in MPO-positive areas normally caused by formation of NETs with bound MPO. While some PLS neutrophils had increased MPO-positive areas after stimulation with glucose oxidase, these cells did not form NETs. The increased MPO-positive areas in these cells were caused by the presence of MPO in decondensated nuclei, as seen in the panels with ×100 magnification. Error bars for the PLS neutrophils demonstrate SD between the 2 experiments performed. One representative experiment out of 3 is shown for quantification of NETosis from the normal controls, and here error bars indicate SD between the 20 images used for quantification. (C) Processing of hCAP-18 by ionomycin-stimulated neutrophils as described in Methods. Cells and medium from ionomycin-stimulated (Io) or unstimulated neutrophils (Us) were blotted with a rabbit antibody against hCAP-18 (34). These functional studies (except the respiratory burst experiment, which was performed only once) were performed on 2 independent occasions 3 months apart with similar results.