Low Elastase Research Articles

Protein C Degradation in vitro by Neutrophil Elastase

Purified protein C is completely degraded into small peptides by in vitro incubation with purified elastase. Protein C is a rather sensitive substrate as degradation is already accomplished by low elastase concentrations (molar enzyme-to-substrate ratio 1:510) and short incubation periods (5 min-60 min). Protein C in a PPSB coagulation factor concentrate is equally degraded and similar split products are detected by blotting techniques. The protein C activity (measured by a chromogenic substrate) is faster reduced by elastase than the protein C concentration (measured by an ELISA). Incubation of normal plasma with high elastase concentrations (5.7 nmol/ml plasma) results in reduction of the protein C band while no split products are detectable. The pathophysiologic significance of the effects of elastase on protein C remains to be elucidated.

Different sensitivity of Pseudomonas aeruginosa exotoxin A and diphtheria toxin to enzymes from polymorphonuclear leukocytes

We demonstrate that exotoxin A (ExoA) of Pseudomonas aeruginosa is one to two orders of magnitude more sensitive than diphtheria toxin (DT) of Corynebacterium diphtheriae to lysosomal enzymes from polymorphonuclear leukocytes (PMN). It is especially sensitive to PMN elastase which inactivates its cell free enzymatic activity and its cytotoxicity as measured with the Chinese hamster ovary cell assay and the rabbit skin test. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed a rapid fragmentation of ExoA into small peptides at low PMN elastase concentrations, whereas DT remained largely uncleaved at PMN elastase concentrations 10 times higher. PMN elastase also removed the cell surface receptors for ExoA and DT on Chinese hamster ovary cells, suggesting that both toxins may be ineffective at local sites of severe inflammation. A comparison of fibroblasts from cystic fibrosis patients and normal healthy individuals revealed no differences in susceptibility to either DT or ExoA; this tends to exclude a genetic defect as an explanation for the absence of ExoA effects in cystic fibrosis patients.

REDUCTION OF DEGRANULATION OF POLYMORPHONUCLEAR LEUKOCYTES BY IMMUNOSUPPRESSION IN PATIENTS FOLLOWING CADAVERIC RENAL TRANSPLANTATION

Plasma levels of main granulocyte components of patients after cadaveric renal transplantation were compared 9 days postoperative with the plasma levels of patients undergoing aortofemoral and iliacofemoral bypass operation or abdominal surgery. Lactoferrin values were significantly lower in patients under immunosuppression with cyclosporine and prednisolone, whereas plasma levels of myeloperoxidase were comparable in all 3 groups of patients. Plasma E-alpha 1 PI values were significantly lower in patients undergoing bypass operation compared to abdominal surgery but did not differ from patients undergoing cadaveric kidney transplantation. Within 22 days postoperatively, there was no difference in the plasma levels of main granulocyte components in patients after kidney transplantation with and without postoperative complications. In vitro incubation of heparinized whole blood and isolated granulocytes obtained from healthy subjects in the presence of CsA, azathioprine, or prednisolone were performed. Only CsA caused inhibition of spontaneous degranulation of polymorphonuclear neutrophils showing significant lower elastase and lactoferrin release. However, in vivo administration of CsA and prednisolone in transplant patients displayed no effect on in vitro degranulation of both whole blood samples and isolated granulocytes. Our data demonstrate that CsA after in vitro incubation inhibits spontaneous granulocyte degranulation but not after in vivo administration. However, in vivo administration of CsA and prednisolone reduces lactoferrin release under certain conditions, e.g., postoperative stress or during hemodialysis therapy.