cathepsin-g and Chediak-Higashi-Syndrome

Neutrophils contain microbicidal proteins packaged in numerous cytoplasmic granules. During phagocytosis, granules fuse to phagolysosomes where the granule contents contribute to the destruction and dissolution of ingested bacteria. The properties of the microbicidal proteins cathepsin G, BPI, and defensins are described and contrasted.

Topics: Animals; Antimicrobial Cationic Peptides; Blood Bactericidal Activity; Blood Proteins; Cathepsin G; Cathepsins; Chediak-Higashi Syndrome; Defensins; Humans; Membrane Proteins; Neutrophils; Serine Endopeptidases

We have reported previously that the abnormally down-regulated protein kinase C (PKC) causes cellular dysfunction observed in natural killer (NK) cells, polymorphonuclear leucocytes (PMNs) and fibroblasts from beige mouse, an animal model of Chediak-Higashi syndrome (CHS). Here we show that the abnormal down-regulation of PKC activity also occurs in Epstein-Barr (EB) virus-transformed cell lines from CHS patients. When CHS cell lines were stimulated with concanavalin A (Con A) for 20 min, the membrane-bound PKC activity declined markedly, whereas that in control cell lines increased. We found that E-64-d, which protects PKC from calpain-mediated proteolysis, reversed the declined PKC activity and corrected the increased Con A cap formation to almost normal levels in CHS cell lines. We confirmed that the dysregulation of PKC activity also occurred in peripheral blood mononuclear leucocytes (PBMC) from CHS patients and that E-64-d corrected both the declined PKC activity and increased Con A cap formation. E-64-d also corrected the reduced lysosomal elastase and cathepsin G activity in CHS cell lines. In contrast, chelerythrin, a specific inhibitor of PKC, and C2-ceramide, which promotes PKC breakdown induced by calpain, increased Con A cap formation and inhibited both elastase and cathepsin G activity in normal cell lines. Moreover, we found that ceramide production in CHS cell lines increased significantly after Con A stimulation, which coincides with our previous observation in fibroblasts from CHS mice. These results suggest an association between ceramide-induced PKC down-regulation and the cellular dysfunctions in CHS.

Topics: Calpain; Cathepsin G; Cathepsins; Cell Line, Transformed; Ceramides; Chediak-Higashi Syndrome; Child; Concanavalin A; Cysteine Proteinase Inhibitors; Down-Regulation; Drug Antagonism; Female; Humans; Leucine; Leukocytes, Mononuclear; Lysosomes; Male; Pancreatic Elastase; Protein Kinase C; Receptor Aggregation; Serine Endopeptidases; Sphingomyelin Phosphodiesterase

We have previously reported that the abnormally rapid down-regulation of protein kinase C (PKC) activity is responsible for the cellular dysfunction in natural killer (NK) cells and polymorphonuclear leukocytes (PMNs) from Chediak-Higashi syndrome (beige) mice. In this report, we examined whether the down-regulation of PKC is associated with giant granule formation in fibroblasts from beige mice. In cultured beige fibroblasts, the membrane-bound PKC activity declined significantly after phorbol ester stimulation. We found that E-64-d, which is a thiol proteinase inhibitor and protects PKC from calpain-mediated proteolysis, reversed the declined PKC activity and prevented giant granule formation in beige fibroblasts. Moreover, E-64-d corrected the reduced lysosomal elastase and cathepsin G activity in beige fibroblasts. In contrast, specific PKC inhibitors, chelerythrin and calphostin C, promoted giant granule formation in normal fibroblasts. We also demonstrate that ceramide production is enhanced in beige fibroblasts and is involved in the rapid down-regulation of PKC. These results suggest that the accelerated breakdown of PKC observed in beige fibroblasts is caused by enhanced ceramide production and is also responsible for giant granule formation.

Topics: Animals; Cathepsin G; Cathepsins; Cells, Cultured; Ceramides; Chediak-Higashi Syndrome; Cysteine Proteinase Inhibitors; Cytoplasmic Granules; Cytosol; Embryo, Mammalian; Fibroblasts; Leucine; Lysosomes; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Pancreatic Elastase; Protein Kinase C; Serine Endopeptidases; Sphingolipids; Tetradecanoylphorbol Acetate

Among other phenotypic defects, the beige mouse is susceptible to infection and has large neutrophil granules that apparently secrete a decreased amount of elastolytic activity. We have shown using in vitro methods that cytosolic inhibitors in beige neutrophils are normal. Although cathepsin G is tightly bound to lysosomal membranes, normal amounts of activity are released in response to degranulating agents. Decreased elastolytic activity is secreted by beige neutrophils because elastase is present in the granules as a 46 kDa proenzyme, which can be activated extracellularly by a protease-dependent mechanism. The current experiments were undertaken to explore the in vivo functions of neutrophils from C57 BI/6J (bg/bg) beige mice using the model of casein-induced acute peritonitis; normal C57 BI/6J (+/+) mice served as controls. The kinetics of neutrophil accumulation in the peritoneum were normal, suggesting normal neutrophil migration. Cathepsin G activity in the cell-free supernatant of peritoneal lavage fluid was normal; elastolytic activity was initially very low but increased to about twice baseline level after 4 h at 25 degrees C and to about 20-fold at 36 h. The appearance of this activity was inhibited to varying degree (54 to 83%) by different protease inhibitors (pepstatin, antipain, aprotinin, leupeptin and chymostatin). We conclude that the decreased amount of elastolytic activity secreted by beige neutrophils into an inflammatory exudate is due to a genetic defect that results in production of a 46 kDa proelastase rather than the normal 29 kDa active elastase; the proelastase can be spontaneously activated by a protease-dependent mechanism. In light of these data, the use of the beige mouse as a model for the Chediak-Higashi syndrome, and as a model in which neutrophils do not produce elastase, must be reconsidered.

Topics: Analysis of Variance; Animals; Ascitic Fluid; Blotting, Western; Caseins; Cathepsin G; Cathepsins; Cell Count; Chediak-Higashi Syndrome; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Leukocyte Elastase; Lysosomes; Male; Mice; Mice, Inbred C57BL; Molecular Weight; Neutrophils; Peritonitis; Protease Inhibitors; Serine Endopeptidases; Species Specificity

Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chédiak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G. There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G G activities. 125I-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-125I-elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G. No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus represent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactivation of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chédiak-Higashi patients to infection.

Topics: Animals; Binding Sites; Bone Marrow; Cathepsin G; Cathepsins; Chediak-Higashi Syndrome; Humans; Mice; Mice, Mutant Strains; Molecular Weight; Neutrophils; Pancreatic Elastase; Serine Endopeptidases

Although several genetic defects are known to impair oxidative microbicidal/cytotoxic mechanisms in human PMN, no deficiencies of PMN granule components that mediate oxygen-independent microbicidal activity have yet been reported. We analyzed PMN from patients with various granulocyte disorders for their content of two azurophil granule constituents, defensins and cathepsin G, that exert microbicidal/cytotoxic activity in vitro, and one component, elastase, that has ancillary microbicidal/cytotoxic activity. PMN from two (of two) patients with specific granule deficiency (SGD) displayed an almost complete deficiency of defensins, which in normal cells constitute greater than 30% of the protein content of azurophil granules. The SGD PMN contained normal or mildly decreased amounts of cathepsin G and elastase. Conversely, the PMN of three (of three) patients with Chediak-Higashi syndrome (CHS) substantially lacked cathepsin G and elastase, but their defensin content was normal or mildly decreased. Both CHS and SGD patients suffer from frequent and severe bacterial infections, and CHS patients frequently develop an atypical lymphoproliferative syndrome. The profound deficiency of PMN components with microbicidal/cytotoxic activity in SGD and CHS may contribute to the clinical manifestations of these disorders.

Topics: Blood Bactericidal Activity; Blood Proteins; Cathepsin G; Cathepsins; Chediak-Higashi Syndrome; Cytoplasmic Granules; Cytotoxins; Defensins; Electrophoresis, Polyacrylamide Gel; Granulomatous Disease, Chronic; Humans; Immunoassay; Neutrophils; Pancreatic Elastase; Peroxidase; Serine Endopeptidases

Elicited peritoneal neutrophils of beige (Chediak-Higashi) mice essentially lack activities of the neutral serine proteinases elastase and cathepsin G, which may explain the increased susceptibility to infection of beige mice and Chediak-Higashi patients. We have examined neutrophils of beige mice at earlier points in their development to determine if the proteinase genes are never expressed or whether they are expressed and then lost during neutrophil maturation. Surprisingly, bone marrow of beige mice had significant elastase and cathepsin G activity (approximately 60% of normal). The results of several experiments indicate that neutrophils were the sole source of elastase and cathepsin G in bone marrow. Neutral proteinase activity was readily demonstrable by histochemical procedures in beige marrow neutrophil precursors up to and including the metamyelocyte stage. However, mature neutrophils of beige marrow had greatly decreased activity. Also mature neutrophils (PMNs) of the peripheral circulation, like peritoneal neutrophils, had very low elastase and cathepsin C activities. Thus we conclude that beige neutrophil precursors express neutral proteinase activity, which is largely and irreversibly depleted by the time they fully mature in marrow.

Topics: Animals; Bone Marrow; Cathepsin G; Cathepsins; Cell Differentiation; Chediak-Higashi Syndrome; Female; Histocytochemistry; Male; Mice; Mice, Inbred C57BL; Neutrophils; Pancreatic Elastase; Peritoneal Cavity; Protease Inhibitors; Serine Endopeptidases

A profound decrease in activities of the two lysosomal serine proteinases, elastase, and cathepsin G, was found in neutrophils of four independent beige mutants. Elastase and cathepsin G activities were assayed with the specific synthetic substrates MeO-Suc-Ala-Ala-Pro-Val-MCA and Suc-Ala-Ala-Pro-Phe-pNA, respectively. The defect is intrinsic to cells of beige mice, since transplantation of bone marrow from normal to mutant mice restored normal proteinase activity, and normal mice transplanted with beige marrow produced neutrophils with a deficiency of proteinase activity. The loss of elastase and cathepsin G activity was confirmed by separation of [3H]diisopropylfluorophosphate-labeled proteins on denaturing gels, which also revealed that other serine proteinases are at normal levels in beige neutrophil extracts. The deficiency of lysosomal proteinase activity appears specific, in that four other common neutrophil lysosomal enzymes, plus the spectrum of major neutrophil proteins are not affected by the beige mutation. The deficiency of proteinase activity is likely not the primary genetic alteration of the beige mutation, since more than one proteinase is affected, and heterozygous F1 mice have normal rather than intermediate levels of both proteinases. The lowered proteinase activity may contribute to the high susceptibility of beige mice and Chediak-Higashi patients to infection.

Topics: Animals; Bone Marrow Transplantation; Cathepsin G; Cathepsins; Chediak-Higashi Syndrome; Heterozygote; Lysosomes; Mice; Mice, Mutant Strains; Neutrophils; Pancreatic Elastase; Peritoneal Cavity; Serine Endopeptidases