Enhanced diacylglycerol production by phospholipase D activation is responsible for abnormal increase in concanavalin A cap formation in polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mice.

We previously reported that enhanced ceramide production induces calpain-mediated proteolysis of protein kinase C (PKC) in leukocytes from Chediak-Higashi syndrome (CHS). In the present study, we demonstrated that phospholipase D (PLD) inhibitors ameliorated abnormal increases in concanavalin A (Con A) cap formation in polymorphonuclear leukocytes (PMNs) from beige mouse, an animal model of CHS. PLD activity in PMNs from beige mice enhanced at 30 to 60s after Con A stimulation. In Con A-stimulated beige PMNs, both neutral sphingomyelinase (N-SMase) and acidic sphingomyelinase (A-SMase) activities enhanced, and ceramide levels are also increased. We found that ceramide levels were reversed by the treatment of beige PMNs with propranolol which inhibits phosphatidic acid phosphohydrolase. In addition, we showed that diacylgycerol (DAG) analogs enhance both N-SMase and A-SMase activities in PMNs from normal mice. We subsequently examined the association of CHS1 with PLD, and showed that expression of a truncated mutant of CHS1 in 293T cells induced abnormally rapid activation of PLD after phorbol ester stimulation. Moreover, we showed that specific inhibitors of 14-3-3 proteins, which interact with CHS1/LYST and bind PKC, did not affect abnormal increases in Con A cap formation in beige PMNs. These results suggest that the enhanced DAG production via the PLD pathway is associated with abnormal increases in Con A cap formation in beige PMNs, and that CHS1 may be involved in the regulation of PLD activity.

A thiol proteinase inhibitor, E-64-d, corrects the abnormalities in concanavalin A cap formation and the lysosomal enzyme activity in leucocytes from patients with Chediak-Higashi syndrome by reversing the down-regulated protein kinase C activity.

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.

Giant granules of neutrophils in Chediak-Higashi syndrome are derived from azurophil granules but not from specific and gelatinase granules.

The abnormal giant granules of Chediak-Higashi syndrome (CHS) neutrophils in humans are thought to be derived from both azurophil and specific granules, whereas the presence of gelatinase granules and their contribution to giant granule formation has not been investigated previously. We have examined the ultrastructure and mobilization of neutrophil granules from a patient with CHS by immunogold electron microscopy and exocytosis experiments of isolated leukocytes. The giant granules contained the azurophil granule components myeloperoxidase and CD63. We found no evidence of involvement of specific or gelatinase granules in the formation of giant granules because lactoferrin and gelatinase were contained in normal-appearing peroxidase-negative granules. On stimulation of leukocytes with N-formyl-methionyl-leucyl-phenylalanine and the calcium ionophore, ionomycin, there was a diminished exocytosis of myeloperoxidase in CHS compared with a healthy control, indicating a lack of mobilization of the giant granules. On the other hand, there was a normal or augmented release of lactoferrin and gelatinase in CHS neutrophils, with gelatinase granules being the most easily mobilized, as known from normal neutrophils. In conclusion, giant granules from CHS neutrophils originate from azurophil granules but not from the specific and gelatinase granules.

Neutrophils in beige mice secrete normal amounts of cathepsin G and a 46 kDa latent form of elastase that can be activated extracellularly by proteolytic activity.

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.

Na,K-ATPase activity in red blood cells from patients with Chediak-Higashi syndrome.

Na,K-ATPase activity of red blood cells from Chediak-Higashi syndrome (CHS) patients and relatives (gene heterozygous) was determined and compared to that of control, healthy, individuals. The enzyme activity was found to be strongly diminished in the CHS patients and slightly lower in their relatives. This reduced activity was due to a lower turnover number of the Na, K-ATPase as well as a decreased number of pumps. The reduced enzyme activity observed in these patients could be the result of an abnormal cell membrane fluidity, and the lowered number of Na, K-pumps could be explained as a consequence of an altered or deficient cell machinery caused by the CHS gene.

Distinguishing between the catalytic potential and apparent expression of tyrosinase activities.

Assays were developed to investigate the catalytic potential and apparent expression of tyrosinase activities. Tyrosine hydroxylase activity determined with cell lysates (in vitro), entire fixed cells (postfixation), or intact living cells (in situ), and 3,4-dihydroxyphenylalanine oxidase assayed spectrophotometrically or by 3,4-dihydroxyphenylalanine staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated the following results: 1) The in situ assay displayed reduced tyrosine hydroxylase activity in all three tyrosinase-positive oculocutaneous albino (OCA) lines except for Chediak-Higashi Syndrome melanocytes, which displayed normal activity; 2) The in vitro assay had comparable activity of tyrosinase-positive OCA melanocytes as controls, except for one tyrosinase-positive OCA cell line, which demonstrated increased activity; 3) The postfixation assay, compared with the in situ assay, had elevated activity (ie. normalization) of tyrosinase in OCA cells but reduced activity in controls; 4) The spectrophotometric assay for 3,4-dihydroxyphenylalanine oxidase activity correlated very well with the tyrosine hydroxylase activity determined by the in vitro assay; 5) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of melanocyte lysates either stained with 3,4-dihydroxyphenylalanine or immunoblotted with anti-tyrosinase detected abnormal tyrosinase bands in the Chediak-Higashi Syndrome and one line of tyrosinase positive OCA melanocytes, and both lines had release of tyrosinase into the growth media. In conclusion, the selection and combination of these tyrosinase assays would be informative for differentiation and characterization of human albinism.

Lysosomal enzyme activities in Chediak-Higashi syndrome: evaluation of lymphoblastoid cell lines and review of the literature.

Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disorder characterized by giant lysosomal granules in all granule-containing cells. Prior examination of lysosomal enzyme activities in granulocytes and other cells derived from patients with CHS have revealed multiple abnormalities, with the predominant finding being diminished activity of many of the enzymes tested. Abnormalities in lysosomal enzyme activity are also found in animal models of CHS (cattle, aleutian mink, and beige mice). In this study, we have examined lymphoblastoid cell lines derived from a patient with CHS and from an individual heterozygous for the CHS gene for acid phosphatase, beta-glucuronidase, and alpha-mannosidase activity. These cell lines have recently been shown to be satisfactory in vitro models for the disease. Acid phosphatase activity was increased in the heterozygous-derived cell line when compared to control while other enzyme activities were normal both in the CHS- and heterozygous-derived cell lines. We have reviewed the literature and summarized published abnormalities of lysosomal enzyme activities in humans and animals with CHS.

Inhibitors of elastase and cathepsin G in Chédiak-Higashi (beige) neutrophils.

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.

The thiol proteinase inhibitors improve the abnormal rapid down-regulation of protein kinase C and the impaired natural killer cell activity in (Chediak-Higashi syndrome) beige mouse.

Protein kinase C (PKC) is essential in intracellular signal transduction for various cell functions including natural killer (NK) cell activity. This enzyme is hydrolysed by calpain, which is Ca2+-dependent thiol proteinase. We showed here that in NK activity-deficient beige (bg/bg) mouse, the model of Chediak-Higashi syndrome, the translocated membrane-bound PKC activity declined rapidly in NK cell-enriched lymphocytes after TPA stimulation. However, the rapid decline was abolished by the pretreatment of cells with leupeptin (a thiol and serine proteinase inhibitor) or E64 (a thiol proteinase inhibitor). Furthermore, these reagents improved the impaired NK cell activity in beige mouse whereas they did not affect NK cell activity in C57BL/6 (+/+) and the heterozygous (+/bg) mice. Meanwhile, TPA stimulation induced only low levels in NK cytotoxic factors (NKCF) release from beige NK cells, but these reagents augmented the lowered NKCF release. These results suggest that the improvement of impaired NK cell activity in beige mouse by the thiol proteinase inhibitors may be due to the elimination of abnormal rapid down-regulation of PKC, resulting in the augmentation of the lowered PKC activity.

Rapid down-regulation of protein kinase C in (Chediak-Higashi syndrome) beige mouse by phorbol ester.

Protein kinase C(PKC) activity in macrophages and polymorphonuclear leukocytes was assayed in beige mouse, the model of Chediak-Higashi syndrome, control C57BL/6 and the heterozygous (+/bg) mice. Regarding enzyme activity in the cytosolic and membrane fractions of these cells, there was no difference between beige mouse and the control. After short-term activation by TPA, the translocated membrane-bound PKC activity in beige mouse decreased rapidly compared with that in control mouse. However, the cytosolic PKC activity decreased at just the same pace as the control. The change in [3H] PDBu binding paralleled the changes in PKC activity. An increase in Ca2+/phospholipid-independent protein kinase by TPA was notable in the membrane fraction of beige mouse. The increase in the kinase activity was abolished and the PKC activity recovered to normal level by the addition of calpain inhibitor, leupeptin, to the incubation of cells along with TPA. Therefore, these findings suggest that a rapid decrease in membrane-bound PKC activity in beige mouse by TPA stimulation is associated with calpain.

Elastase and cathepsin G activities are present in immature bone marrow neutrophils and absent in late marrow and circulating neutrophils of beige (Chediak-Higashi) mice.

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.

Normal-sized primary lysosomes are present in Chediak-Higashi syndrome neutrophils.

Azurophil granules of normal neutrophils are known to be primary lysosomes and contain myeloperoxidase activity. The present study has used ultrastructural cytochemistry to selectively stain myeloperoxidase containing granules of normal and Chediak-Higashi syndrome neutrophils and serial thin sections to determine if all peroxidase-positive organelles in Chediak-Higashi syndrome cells are protrusions of the huge inclusions characteristic of the disorder. Peroxidase-positive organelles in polymorphonuclear leukocytes from three patients with Chediak-Higashi syndrome varied in size from vesicles and normal-sized lysosomes to the huge bodies filling the cytoplasm. Serial sections demonstrated that the small and normal-sized organelles were commonly present and independent of the giant granules. One type of the normal-sized lysosomes contained small vesicles free of peroxidase activity, conveying a honeycomb-like appearance to the matrix. This variety, in particular, was identified in various stages of fusion with giant organelles in circulating Chediak-Higashi syndrome neutrophils.

Characterization of lysosomes and lysosomal enzymes from Chediak-Higashi-syndrome cultured fibroblasts.

Chediak-Higashi-syndrome cultured skin fibroblasts were used to study the possible involvement of lysosomal enzymes and lysosomal dysfunction in this disorder. Our evidence indicated that Chediak-Higashi fibroblasts displayed a significant decrease in the specific activity of the acidic alpha-D-mannosidase (pH 4.2) compared with normal controls. Additional studies revealed a small, but significant, decrease in the rate of degradation of 125I-labelled beta-D-glucosidase that had been endocytosed into Chediak-Higashi cells.

Lysosomal elastase and cathepsin G in beige mice. Neutrophils of beige (Chediak-Higashi) mice selectively lack lysosomal elastase and cathepsin G.

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.

Peroxidase activity in salivary glands and saliva of beige (Chediak-Higashi) mice.

Peroxidase activity was lower in the submandibular and parotid glands of beige mice than in controls. A lower level of activity was also observed in the isoproterenol-elicited saliva of beige mice.

Production of plasminogen activator by human natural killer cells. Large granular lymphocytes.

In this report we have used highly purified populations of natural killer (NK) cells: large granular lymphocytes (LGL). This study demonstrates that freshly isolated and interleukin 2-cultured LGL produce the specific neutral serine protease, plasminogen activator (PA). We have found that the enzyme is expressed in both an extracellular form as well as in a cell-associated form. Upon subcellular distribution the latter form of the enzyme is associated with a cell-surface membrane-enriched fraction. LGL PA exists in multiple molecular weight forms ranging from 100,000 to 26,000. Interferon (IFN), the major positive regulator of NK cytolytic activity, caused a substantial enhancement of cell-associated, but not extracellular, PA. In contrast, LGL isolated from patients with Chediak-Higashi syndrome, who are known to be defective in NK activity, displayed low PA activity, altered morphology, and low NK killing relative to LGL isolated from normal donors. The possible role of LGL PA in the lysis of tumor cells by NK cells, either directly or indirectly, is discussed.