concanavalin-a and Chediak-Higashi-Syndrome

Topics: Animals; Ascorbic Acid; Chediak-Higashi Syndrome; Concanavalin A; Cyclic GMP; Disease Models, Animal; Humans; Immunity, Cellular; Leukocytes; Lupus Erythematosus, Systemic; Mice; Mice, Inbred NZB; Prostaglandins

The Chediak-Higashi (CH) syndrome of man and several animal species is characterized by the presence of abnormal giant granules in all granule-containing cells and by defects in chemotaxis and lysosomal degranulation during phagocytosis in polymorphonuclear leukocytes (PMNs). Since similar functional abnormalities have been reported in normal PMNs following exposure to colchicine and other agents that disrupt microtubles it was proposed that microtubule function may be impaired in the CH syndrome. The mobility of concanavalin A (con A)-receptor complexes on PMN membranes was used to test microtubule integrity. Normal PMNs showed a uniform distribution of membrane-bound con A. By contrast, con A was aggregated into surface caps on both colchicine-treated normal PMNs and untreated PMNs from mice and a patient with CH syndrome. This result is consistent with impaired microtubule function in the CH cells. The spontaneous capping response of CH PMNs was inhibited by cyclic GMP and by cholinergic agonists that can elevate cyclic GMP levels in neutrophils. This raised the possibility that the microtubule defect in CH cells may be correctable by treatments that increase cyclic GMP generation. Direct evidence for both the absence of microtubule assembly in con A-treated PMNs from the CH patient and for normal microtubule assembly in CH PMNs incubated with cyclic GMP and cholinergic agonists prior to con A treatment was obtained by electron microscopy. In addition, evidence for a direct relationship between the microtubule defect and the development of giant lysosomes in CH cells was obtained. Thus, CH fibroblasts grown in vitro developed abnormal lysosomes in the majority of cells. However, the same cells cultured in the presence of cholinergic agonists developed a majority of lysosomes that were morphologically normal at the level of the light microscope. Similarly, granule morphology appeared normal in peripheral blood leukocytes from mice treated chronically in vivo with cholinergic agonists.

Topics: Animals; Chediak-Higashi Syndrome; Chemotaxis; Colchicine; Concanavalin A; Cyclic AMP; Cyclic GMP; Cytoplasmic Granules; Glycoproteins; Humans; Mice; Microtubules; Parasympatholytics; Phagocytosis; Receptors, Drug

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.

Topics: Animals; Cells, Cultured; Ceramides; Chediak-Higashi Syndrome; Concanavalin A; Diglycerides; Disease Models, Animal; Enzyme Activation; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neutrophils; Phospholipase D; Protein Kinase C; Receptor Aggregation; Sequence Deletion; Sphingomyelin Phosphodiesterase; Vesicular Transport Proteins

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

Protein kinase C (PKC) plays an essential role in intracellular signal transduction for various cell functions, including concanavalin A (Con A)-induced cap formation. This enzyme is known to be proteolysed by calpain, which is a Ca2(+)-dependent thiol proteinase. As reported previously, in polymorphonuclear leukocytes (PMNs) from beige mouse, the model of Chediak-Higashi syndrome, Con A-induced cap formation significantly increased compared with that in normal mouse. However, after pretreatment of beige PMNs with the thiol proteinase inhibitors leupeptin or E-64, the capping decreased to normal levels. Meanwhile, Con A-induced the translocation of PKC from the cytosolic to membrane fraction within 5 min in both mice, which is essential to the activation of this enzyme. However, after the translocation, an abnormal rapid decline in membrane-bound PKC activity was noted in beige mouse PMNs. Both leupeptin and E-64 also corrected the rapid decline in PKC activity observed in the beige mouse. These findings suggest that the normalization of Con A cap formation in beige mouse PMNs by the thiol proteinase inhibitors is associated with the correction of abnormality in PKC activity.

Topics: Animals; Calpain; Chediak-Higashi Syndrome; Concanavalin A; Disease Models, Animal; Down-Regulation; Immunologic Capping; Mice; Mice, Inbred C57BL; Neutrophils; Protein Kinase C

Peripheral blood lymphocytes from two Chediak-Higashi syndrome (CHS) patients were examined for their 1) natural killer (NK) cell functions 2) concanavalin A (Con A)-inducible suppressor cell activity, 3) soluble suppressor factor production, and 4) responsiveness to interferon alpha and interleukin-2 in comparison with age-matched normal controls. Peripheral blood lymphocytes or NK-enriched large granular lymphocytes from Chediak-Higashi syndrome patients showed negligible cytotoxic activity against several target cells. Although the NK activity of Chediak-Higashi syndrome lymphocytes could not be restored to normal levels by treatment with either interferon or interleukin-2, the percent enhancement of NK activity was higher for the patients than the controls. Soluble suppressor factor activity of culture supernates from the lymphocytes of Chediak-Higashi syndrome patients significantly inhibited the NK activity of allogeneic, normal peripheral blood lymphocytes, whereas lymphocytes from Chediak-Higashi syndrome patients precultured with Con A failed to suppress the cytotoxic activity of normal lymphocytes. These results demonstrate a previously unrecognized suppressor cell dysfunction in CHS patients.

Topics: Chediak-Higashi Syndrome; Child; Concanavalin A; Cytotoxicity, Immunologic; Female; Humans; Immunity, Cellular; Immunity, Innate; Interferons; Interleukin-2; Killer Cells, Natural; Lymphocytes; Lysosomes; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory

Topics: Adult; Arthritis, Rheumatoid; Ascorbic Acid; B-Lymphocytes; Chediak-Higashi Syndrome; Concanavalin A; Cyclic GMP; Humans; Hypersensitivity, Delayed; Immunity, Cellular; Immunoglobulins; In Vitro Techniques; Lymphocytes; Middle Aged; Monocytes; Pokeweed Mitogens; T-Lymphocytes, Regulatory

It was previously shown that the abnormal surface characteristics and defective bactericidal function of polymorphonuclear leucocytes (PMN) in the Chediak-Higashi syndrome (CHS) are correlated with impaired microtubule assembly, and in one patient direct electron microscopic evidence for an anomaly in microtubule assembly following surface membrane activation by concanavalin A (Con A). We show that very few microtubules are visible in CHS leucocytes from two additional patients under conditions where normal PMNs contain abundant microtubules, and that both in vivo and in vitro exposure of the CHS leucocytes to ascorbic acid promotes the assembly of microtubules. This agent, which normalizes chemotaxis and degranulation in CHS leucocytes, is shown also to correct granulocyte adherence in these leucocytes. It is suggested that the improved clinical course of patients with CHS following treatment with ascorbic acid is related at least in part to improvement of microtubule assembly and PMN function by the ascorbic acid.

Topics: Ascorbic Acid; Cell Adhesion; Chediak-Higashi Syndrome; Child, Preschool; Concanavalin A; Female; Humans; Immunologic Capping; Infant; Microscopy, Electron; Microtubules; Neutrophils

Chediak-Higashi (CH) syndrome is a genetic disorder of children and certain animal species including the beige mouse. We have previously described a membrane abnormality in CH mouse polymorphonuclear leukocytes (PMH). Whereas normal mouse PMN do not form surface caps with concanavalin A except after treatment with agents such as colchicine that inhibit microtubule assembly, CH mouse PMN show spontaneous cap formation. This capping is inhibited by 3',5 cyclic guanosine monophosphate and by the cholinergic agonists carbamylcholine and carbamyl beta-methylcholine that increase 3',5' cyclic guanosine monophosphate generation. These data suggested that microtubule function may be impaired in CH syndrome perhaps secondary to an abnormality in 3',5' cyclic guanosine monophosphate generation. The cholinergic agonists were also shown to prevent development of the giant granules that are pathognomonic of CH syndrome in embryonic fibroblasts isolated from CH mice and cultured in vitro. In this report it is shown that an extreme degree of spontaneous concanavalin A cap formation is also characteristic of peripheral blood PMN from two patients with CH syndrome. This indicates an abnormality of microtubule function in CH syndrome in man. 3',5' cyclic guanosine monophasphate, carbamylcholine, and carbamyl beta-methylcholine reduce spontaneous capping in CH cells. In addition, it is shown that monocytes isolated from the patients' blood and incubated in tissue culture generate a large complement of abnormal granules. When the same cells mature in vitro in the presence of carbamylcholine or carbamyl beta-methylcholine, the proportion of cells containing morphologically normal granules is significantly increased. These responses can be reproduced in vivo in the beige (CH) mouse. Animals treated for 3 wk and longer with carbamylcholine or carbamyl beta-methylcholline show normal granule morphology and a normal degree of concanavalin A cap formation in peripheral blood PMN leukocytes.

Topics: Adult; Animals; Binding Sites; Cells, Cultured; Chediak-Higashi Syndrome; Child, Preschool; Concanavalin A; Cytoplasmic Granules; Disease Models, Animal; Humans; Leukocytes; Male; Methacholine Compounds; Mice; Mice, Inbred Strains; Microscopy, Fluorescence; Microtubules

Topics: Animals; Binding Sites, Antibody; Cell Membrane; Chediak-Higashi Syndrome; Colchicine; Concanavalin A; Cyclic GMP; Disease Models, Animal; Leukocytes; Mice