Effect of E. faecalis on the release of serine proteases elastase and cathepsin G, and collagenase‐2 (MMP‐8) by human polymorphonuclear leukocytes (PMNs)

Abstract

To measure the release of hydrolytic enzymes [elastase, cathepsin G and collagenase-2 (MMP-8)] from human polymorphonuclear leukocytes (PMNs) during interaction with strains of Enterococcus faecalis isolated from endodontic infections. Six E. faecalis strains isolated from treatment resistant cases of apical periodontitis were included in the study. Overnight cultures of the microbes were used for the experiments. PMNs were isolated using the Ficoll Paque technique, and their vitality was assessed throughout the experiments by the Trypan Blue exclusion test. A known amount of microbes and PMNs were mixed in PBS at +37 degrees C in air, and the release of elastase, cathepsin G and MMP-8 was measured at 0, 20, 60 and 120 min after initiation of incubation. The activities of elastase and cathepsin G were analysed by spectrophotometer assays using specific synthetic peptide substrates, and MMP-8 by western immunoblotting quantitated by computer densitometry. PMNs incubated in buffer without any added microbes served as negative controls, cells incubated with 5 ng mL(-1) phorbol myristic acetate (PMA) served as a positive control. The 95% confidence interval was used to compare the relative amount of elastase and cathepsin G released from the samples. One E. faecalis strain induced a similar or higher elastase, cathepsin G and MMP-8 release than the positive control, whereas the other five strains induced only moderate or no release of the three enzymes examined as compared with the negative and positive controls. Western immunoblot revealed that released MMP-8 had molecular sizes of 60 and 75 kDa representing active and latent forms of MMP-8. In addition, >110 kDa high molecular size and a fragmented 20-30 kDa MMP-8 species could be observed. The majority of the E. faecalis strains induced little or no release of hydrolytic enzymes from the PMN cells. The finding may partly explain the clinical observation that root canal infections dominated by E. faecalis are usually symptom free.