Role of polymorphonuclear leukocyte-derived serine proteinases in defense against Actinobacillus actinomycetemcomitans

Abstract

Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such as Actinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense against A. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity against A. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to kill A. actinomycetemcomitans in an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.

Figures

FIG. 1.

Degradation of leukotoxin by PMNs and cytotoxic action of leukotoxin. (A) Western blot detection of cleavage of leukotoxin by PMN lysates of family A and family B. Aliquots of 150 ng of leukotoxin were incubated with equal protein amounts (2 μg) of PMN cell lysates for 3 h at 37°C. As a control, an equal quantity of leukotoxin was incubated in assay buffer (left lane, L) or incubated with PMN lysates isolated from a healthy donor (H1). (B) Western blot detection of leukotoxin in supernatants after a 1-h incubation of leukotoxin at 150 ng with PMNs (1.8 × 106 cells) isolated from family A and family C at 37°C. Proteins in equal volumes of supernatants were separated by SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. Leukotoxin and fragments were detected with polyclonal antibodies raised against the leukotoxin. As a control, 150 ng of leukotoxin was incubated in assay buffer (right lane, L). A sample of a healthy individual (H2) was also included. Family A: parents AC1 and AC2 and patients AP1 and AP2. Family B: parents BC1 and BC2 and patient BP1. Family C: parents CC1 and CC2 and patient CP1. (C) On the left side, two micrographs are shown of PMNs isolated from a parent of family A (AC1); on the right, two micrographs are shown of PMNs isolated from a PLS patient of family A (AP1). The top micrographs show PMNs incubated in assay buffer for 1 h; the bottom micrographs show PMNs incubated in the presence of 150 ng of leukotoxin. Note the absence of discernible organelles in the patient's PMNs upon exposure to leukotoxin. (D) Histomorphometric analysis of the percentage of “nonviable” cells compared to the total number of cells. Controls, n = 4; patients, n = 4. The mean ± the standard deviation is given. *, P < 0.05 (Mann-Whitney test).

FIG. 2.

Immunoblot of hCAP-18/LL-37 of exocytosed material of PMNs isolated from PLS patients and their parents from family A. (A) Equal volumes (10 μl) of exocytosed material were analyzed. PMNs were stimulated with PMA (4 μg/ml), fMLP (2.5 μM), or ionomycin (1 μM) for 15 min at 37°C. Supernatants were analyzed by Western blotting with monoclonal antibodies to LL-37. hCAP-18 and LL-37 are indicated by arrows. Intermediate fragments indicate partially processed hCAP-18. The lower panel is the lower half of the immunoblot shown in the upper panel with a longer exposure time. Parents are indicates by AC1 and AC2; patients are indicated by AP1 and AP2. All samples were run on one gel. (B) Proteinase 3 activity in ionomycin exocytosed material of PMNs of family A. Samples (10 μl) were analyzed for proteinase 3 activity. Activity is expressed as the increase in absorption at 405 nm over time. Blanc, sample consisting of the reaction mixture was included in this assay to correct for spontaneous decay of the substrate. Note that the lines of the patients and the blanc coincide. The family A parents are indicated by AC1 and AC2; the family A patients are indicated by AP1 and AP2.

FIG. 3.

Anaerobic killing of A. actinomycetemcomitans by PMNs. PMNs isolated from parents and patients of family A were incubated with A. actinomycetemcomitans (ratio 1:10) at 37°C for 60 or 120 min under anaerobic conditions. Serial dilutions of cell lysate samples were plated on blood agar plates and cultured for 48 h, and the number of CFU was counted. Columns indicate the mean percentage ± the SD of surviving CFU as a measure for the number of bacteria killed. Parents, n = 2; patients, n = 2. Due to the small sample number in the groups, the differences were not compared statistically.