A family with Papillon-Lefèvre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity

Abstract

IntroductionNatural killer (NK) cells are cytolytic lymphocytes whose function is to control infection and malignancy. Granule exocytosis delivers granzymes, a family of pro-apoptotic serine proteases, to target cells leading to the induction of caspasedependent and caspase-independent apoptosis. 1 Mouse granzyme B is required for the rapid induction of apoptosis in target cells. 2 However, other granzyme molecules contribute to the overall cytolytic phenotype, 3 with human cells expressing granzymes A, B, H, K, and M. 1,4 Activation of granzymes requires the removal of an N-terminal dipeptide. 5 Cathepsin C, a cysteine protease also known as dipeptidyl peptidase I (DPPI), has been implicated in granzyme A and B processing. 6,7 Cathepsin C-deficient mice fail to process granzymes A and B and hence lack both NK and T-cell-mediated cytolytic activity. 7 Human cathepsin C deficiency is the cause of Papillon-Lefèvre syndrome (PLS). [8][9][10] This is an extremely rare, autosomal recessive condition characterized by palmoplantar keratosis and severe periodontitis. In addition, skin infections and liver abscesses have been reported in PLS, 10,11 strongly suggesting an immunodeficiency component to the disease. However, in contrast to the mouse model, interleukin-2 (IL-2)-generated lymphokine activated killer (LAK) cell activity in these patients appears to be normal, and granzyme B is active in PLS LAK cells. 12 Here, we have tested the requirement for cathepsin C in the processing of granzyme B in unstimulated human NK cells from PLS patients. We describe an NK cell cytolytic defect and a failure of granzyme B activation. Study design PatientsPLS patients 1 and 2 are brother and sister from a consanguineous family, family 6 in Toomes et al. 8 Both patients are homozygous for a mutation changing glycine 277 of cathepsin C to a serine (G277S). This nomenclature is that used in the structural study 13 and refers to cathepsin C with the 24 amino-acid signal peptide removed. A third family member is unaffected by PLS and has one G277S allele and one wild-type allele and is referred to as the heterozygote. Blood samples from patients, the heterozygote, and healthy control donors were collected with informed consent and approval from the Leeds Teaching Hospital NHS Trust UK ethics committee. Functional studiesNK cells were phenotyped in whole blood using flow cytometry and isolated from peripheral blood mononuclear cells (PBMCs) using the NK isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were used directly or cultured for 10 to 14 days with 50 units/mL human recombinant IL-2 (Sigma, St Louis, MO). Whole cell lysates were assayed for cathepsin C or granzyme B activity using the colorimetric substrates Gly-Phe-pNA (Sigma) and Ac-IEPD-pNA (Calbiochem, San Diego, CA), respectively, 14,15 or used in immunoblotting experiments. Aprotinin agarose binding was performed as described. 16,17 Killing and caspase activation assays were performed using flow cytometry, using CellTracker dye (Molecular Probes, Eug...

Proteolytic Activation of the Cytotoxic Phenotype during Human NK Cell Development

“…For killing assays, membrane integrity was assessed by propidium iodide staining and caspase activation was analyzed using PhiPhiLux (Calbiochem) staining, as previously described (8). Killing assays were performed at least twice, using NK cells differentiated from separate cord blood samples.…”

“…Cathepsin C activity and aspartase (aspase) activity (as a measure of granzyme B activity) were determined by measuring hydrolysis of the synthetic substrates GF-pNA (Sigma-Aldrich) and AcIEPD-pNA (Calbiochem) respectively, as previously described (8,26). Inhibition of cathepsin C activity with 10 M Gly-Phe-DMK (27) (MP Biomedicals) was performed for 4 or 16 h at 37°C before washing and lysis of cells; cells were also treated with DMSO alone as a solvent control (0.1% v/v), for 16 h. Perforin immunoblots and inhibition of perforin processing with 10 mM NH 4 Cl were performed, as described (14), using the 2d4-perf Ab, provided by G. Griffiths (Cambridge Institute for Medical Research, U.K.).…”

“…The cysteine protease cathepsin C has been implicated in granzyme processing and is sufficient to remove the N-terminal prodomain from granzymes A, B, H, and K in vitro (3)(4)(5)(6). Furthermore, cathepsin C-deficient mice (7) and humans (8,9) show reduced NK cell cytotoxic activity associated with a failure to remove the granzyme B N-terminal prodomain (7,8). However, cathepsin C-deficient mice and humans can use an alternative (but as yet unidentified) enzyme to activate granzymes (7,8,10,11).…”

“…Furthermore, cathepsin C-deficient mice (7) and humans (8,9) show reduced NK cell cytotoxic activity associated with a failure to remove the granzyme B N-terminal prodomain (7,8). However, cathepsin C-deficient mice and humans can use an alternative (but as yet unidentified) enzyme to activate granzymes (7,8,10,11). Cathepsin C itself is synthesized as a zymogen and also requires proteolytic cleavage for activation (12).…”

Syndromes That Include Both Palmoplantar Keratoderma And Severe Periodontitis: A Review

“…PMNs chemotaxis, phagocytosis of opsonized Staphylococcus aureus, and production of superoxide radicals by PMNs are significantly impaired in PLS patients [24]. These functionally defective PMNs are reported to be inheritable, thus leading to difficulty in coping with periodontitis-associated pathogens [25,26].…”