Myeloperoxidase: its structure and expression during myeloid differentiation

Myeloperoxidase (MPO) is a major protein present in myeloid cells and is used by these cells to help kill microbes. The human promyelocytic HL-60 line can be induced to differentiate to granulocytes or macrophagelike cells. Poly (A) containing RNA was isolated from HL-60 granulocytes, HL-60 macrophages, HL-60 blasts, and normal human granulocytes. The mRNA was translated in a reticulocyte lysate system in the presence of 35S-methionine. The MPO was precipitated from the lysate with rabbit IgG antiserum to human MPO. The resulting precipitate from HL-60 blasts gave a major band of radioactivity of approximately 77,000 daltons and another band at approximately 46,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The MPO identity of the labeled bands was confirmed by cold competition. The relative mRNA activity expressed as a percentage of radioactivity incorporated into MPO (77,000-dalton band) as compared with total trichloracetic acid (TCA) precipitable radioactivity was 0.2%. Negligible mRNA activity for MPO was present in HL-60 granulocytes, HL-60 macrophages, and normal human granulocytes. Pulse- chase experiments showed that MPO was an approximate 75,000-dalton major band and 77,000-dalton minor band of radioactivity after HL-60 blasts were labeled for 1/2 hour with 35S-methionine and the cell lysate immunoprecipitated and subjected to SDS-PAGE. The chase experiments (one to 24 hours) showed that the 77,000- and 75,000-dalton bands of radioactivity were replaced with two major bands (55,000 and 15,000 daltons) and one minor band (approximately 39,000 daltons) of radioactivity. Six-hour 35S-methionine labeling experiments showed that the relative rate of MPO synthesis compared with total TCA precipitable radioactivity was 0.5% in HL-60 blasts and almost negligible in HL-60 macrophages and granulocytes, normal human granulocytes, and B- lymphocytes. The KG-1 myeloblasts and KG-1a early myeloblasts synthesized a small amount of the 75,000-dalton MPO protein. Although HL-60 cells no longer synthesized MPO after differentiation, HL-60 granulocytes and HL-60 macrophages continued to contain MPO as measured by enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aberrant restriction endonuclease digests of DNA from subjects with hereditary myeloperoxidase deficiency

Myeloperoxidase (MPO) is a critical component in the oxygen-dependent microbicidal activity of neutrophils. Hereditary deficiency of MPO occurs commonly, but its genetic basis has not been determined. Previously we have reported the presence of an 89-kilodalton protein, likely pro-MPO, in normal and MPO-deficient neutrophils and hypothesized that the absence of peroxidase activity in neutrophils from affected subjects was the result of defective posttranslational processing of pro-MPO. In this study we analyzed nucleic acids from three completely and two partially MPO-deficient individuals by using a cDNA probe for MPO. The affected individuals studied are unrelated to one another. Neutrophils from all affected subjects lacked mature MPO subunits; however, a monospecific antibody for MPO identified in these cells a high-molecular weight protein that is the same size as pro-MPO. Northern blots demonstrated that the amount and size of RNA (3.3 kilobases [kb]) in a completely deficient subject was normal. BglII digests of genomic DNA from control individuals (n = 14) contained three fragments that hybridized with cDNA for MPO under very stringent conditions. In contrast, BglII digests of genomic DNA from completely MPO-deficient individuals contained an extra fragment of 2.1 kb that was not present in DNA from controls. In addition, two different endonuclease digest patterns were found in MPO-deficient subjects who were biochemically and phenotypically identical. We conclude from these studies that (a) hereditary MPO deficiency is not associated with a major deletion or rearrangement of the MPO gene; (b) myeloid precursors in an MPO-deficient individual contain normal amounts of an mRNA that is the same size as that for MPO in normal individuals; and (c) the genetic basis for MPO deficiency may be heterogeneous, with at least two genotypes generating the same phenotype. These findings are consistent with the hypothesis that the genetic defect in MPO deficiency results in synthesis of a modified pro-MPO that undergoes defective posttranslational processing.     

Myeloperoxidase biosynthesis by a human promyelocytic leukemia cell line: insight into myeloperoxidase deficiency

The biosynthesis and processing of myeloperoxidase (MPO), a cationic enzyme present in the azurophilic granules of human polymorphonuclear leukocytes (PMNs), were studied in the human promyelocytic leukemia cell line, HL-60. HL-60 cells produce large quantities of enzymatically active MPO that has the same electrophoretic behavior as MPO isolated from normal PMNs. Mature MPO is a glycoprotein of approximately 150,000 molecular weight (mol wt) composed of two heavy-light protomers (alpha 2 beta 2) with subunits of 59,000 and 13,500 mol wt, respectively, under reducing conditions. The primary translation product of MPO messenger RNA (mRNA) isolated from HL-60 cells was a single polypeptide of mol wt 80,000. In HL-60 cells labeled with [35S]-methionine, the labeled MPO isolated by immunoprecipitation had a mol wt of 89,000. Treatment of this 89-kilodalton (kDa) species with endoglycosidase H produced a 79-kDa peptide, suggesting that the 89-kDa protein contained high-mannose side chains. The 89-kDa species had no detectable peroxidase activity. During chase experiments some of the 89-kDa peptide was processed to smaller species of mol wt 39,000, 59,000, and 13,500, although a fraction of the 89-kDa peptide remained unprocessed after a chase of 100 hours. In addition, a small amount of the 89-kDa peptide appeared in the medium without any of the processed smaller peptides. These studies suggest that the primary translation product in MPO biosynthesis is an 80-kDa peptide that undergoes cotranslational cleavage of the signal peptide and glycosylation to produce an 89-kDa pro-MPO, that pro-MPO is a single polypeptide containing the alpha and beta subunits of MPO and contains endoglycosidase H-susceptible high- mannose side chains, and that posttranslational modification of pro-MPO results in targeting to the lysosome and proteolytic maturation of pro- MPO to active enzyme. In light of the previous observation that MPO- deficient and normal PMNs contain an 89-kDa protein immunochemically related to MPO, these studies on MPO biosynthesis indirectly support the hypothesis that defective posttranslation processing by pro-MPO may underlie hereditary MPO deficiency.     

Myeloperoxidase: a myeloid cell nuclear antigen with DNA-binding properties.

An antigen histochemically localized in the nuclei and cytoplasmic granules of normal and leukemic human myeloid cells has been identified as myeloperoxidase (MPO; EC 1.11.1.7). The localization and amount of the enzyme was determined by using a murine monoclonal antibody designated H-43-5 raised against nuclear proteins derived from human promyelocytic HL-60 leukemia cells. The highest amount of nuclear MPO (3.5 micrograms per 10(6) nuclei) was found in granulocytes; less than half of this amount was detected in nuclei from HL-60 cells. Still lower levels were found in nuclei from monocytes and a series of human monomyelocytic leukemia cells. MPO from HL-60 cells was purified by immunoaffinity chromatography and fractionated into three components (forms I, II, and III) by CM-cellulose chromatography. Chromatography of these MPO forms on DNA-Sepharose columns confirmed that all three forms of MPO were tightly bound to DNA with apparent relative affinities in the order of form III greater than form II greater than form I. The affinity of MPO form III for DNA was sufficient to enable the formation and elution of DNA-MPO complexes during size-exclusion chromatography at high ionic strength and neutral pH. This form of MPO was also able to shield DNA from strand scission induced by active oxygen species generated by xanthine oxidase acting aerobically on xanthine. These data suggest that intranuclear MPO may help to protect DNA against damage resulting from oxygen radicals produced during myeloid cell maturation and function.     

Cloning and functional characterization of GMPR2, a novel human guanosine monophosphate reductase,which promotes the monocytic differentiation of HL-60 leukemia cells

PURPOSE: To identify the biological function of a novel molecule which shows high homology with human guanosine monophosphate reductase (GMPR) and is named GMPR2. METHODS: GMPR2 cDNA was cloned from the cDNA library of human dendritic cells and was characterized by Bioinformatics. The expression pattern of GMPR2 was analyzed by Northern blotting. The enzymatic activity of the purified recombinant GMPR2 protein was determined using a spectrophotometric assay. HL-60 leukemia cells were transfected with GMPR2 and the expression of CD14 and myeloperoxidase (MPO) in HL-60 cells with and without 12- o-tetra-decanoyl-phorbol-13-acetate (TPA) induction was monitored by FACS analysis. RESULTS: The novel gene contained ten exons and nine introns and was mapped to 14q11-21. Northern blotting indicated a ubiquitous expression of GMPR2 mRNA in most of the human tissues and cancer cell lines investigated. The recombinant GMPR2 protein was able to reduce GMP. The expression of CD14 and MPO in HL-60 leukemia cells overexpressing GMPR2 clearly increased after induction by TPA. CONCLUSIONS: GMPR2 is a novel human GMP reductase, and overexpression of GMPR2 can promote the monocytic differentiation of HL-60 leukemia cells.     

Myeloperoxidase-deficient polymorphonuclear leucocytes. (I) Incidence in untreated myeloid leukaemia, lymphoid leukaemia and normal humans

Neutrophil myeloperoxidase (MPO) activity was analysed semi-quantitatively both by (i) MPO-scoring of polymorphonuclear leucocytes (PMN) and (ii) counting the MPO-deficient PMN (PMN lacking MPO) in 164 subjects (60 cases of leukaemia and 104 normal humans). The scoring method showed that 10 out of 21 (48%) cases of acute myeloid leukaemia (AML), 2 out of 10 (20%) cases of chronic myeloid leukaemia (CML), 0 out of 29 cases of lymphoid leukaemia (ALL + CLL), and 1 out of 104 normal humans had decreased MPO scores. These figures correlated well with the more simple counting of PMN lacking MPO in the same groups: 8 out of 21 (37%) cases of AML, 6 out of 10 (60%) cases of CML and 0 out of 29 cases of lymphoid leukaemia showing more than 4% PMN lacking MPO. In cases of otherwise unclassifiable acute leukaemia, a decreased MPO score and an increased number of MPO-deficient PMN suggests the diagnosis of AML and not ALL. Counting the number of PMN lacking MPO was found to be a time-saving and even more reliable method than the semiquantitative scoring of MPO activity in PMN.     

Myeloperoxidase-Deficient Polymorphonuclear Leucocytes

Neutrophil myeloperoxidase (MPO) activity was analysed semi-quantitatively both by (i) MPO-scoring of polymorphonuclear leucocytes (PMN) and (ii) counting the MPO-deficient PMN (PMN lacking MPO) in 164 subjects (60 cases of leukaemia and 104 normal humans). The scoring method showed that 10 out of 21 (48%) cases of acute myeloid leukaemia (AML), 2 out of 10 (20 %) cases of chronic myeloid leukaemia (CML), 0 out of 29 cases of lymphoid leukaemia (ALL + CLL), and 1 out of 104 normal humans had decreased MPO scores. These figures correlated well with the more simple counting of PMN lacking MPO in the same groups: 8 out of 21 (37 %) cases of AML, 6 out of 10 (60 %) cases of CML and 0 out of 29 cases of lymphoid leukaemia showing more than 4 % PMN lacking MPO. In cases of otherwise unclassifiable acute leukaemia, a decreased MPO score and an increased number of MPO-deficient PMN suggests the diagnosis of AML and not ALL. Counting the number of PMN lacking MPO was found to be a time-saving and even more reliable method than the semiquantitative scoring of MPO activity in PMN.     

Biosynthesis and processing of myeloperoxidase--a marker for myeloid cell differentiation

Myeloperoxidase (MPO), a heme protein, is a major component of azurophilic granules of neutrophils. Optimal oxygen-dependent microbicidal activity depends on MPO as the critical enzyme for the generation of hypochlorous acid and other toxic oxygen products. MPO is synthesized during the promyelocytic stage of myeloid differentiation, the stage at which azurophilic granules are formed. Like other lysosomal enzymes, MPO is synthesized as a larger precursor which is subsequently processed and transported intracellularly to the lysosomes. The primary translation product is a single 80-kDa protein which undergoes cotranslational N-linked glycosylation to produce a 92-kDa glycoprotein. Glucosidases in the endoplasmic reticulum or early cis Golgi convert the proMPO to a 90-kDa form which is sorted into a prelysosomal compartment that undergoes final proteolytic maturation to native MPO, a pair of heavy-light protomers with subunits of 60 kDa and a 12 kDa. These events contrast with similar processes seen with other lysosomal enzymes in two ways. First, alkalinization of lysosomes with NH4+ does not alter processing or transport, in contrast to the pH dependence of these processes for other lysosomal enzymes. However, some studies indicate retardation of processing in the presence of the proton ionophore monensin. Second, intracellular transport of MPO is not apparently mediated by the mannose-6-phosphate receptor system. The gene for MPO is on the long arm of chromosome 17 (17q22, 23) near the breakpoint of the 15, 17 translocation of acute promyelocytic leukemia. The gene spans approximately 14 kb and contains 11 irons and 12 exons. The cloned full-length cDNA is approximately 2.2 kb and both normal bone marrow and cultured promyelocytic leukemia cells express two species of mRNA. Inherited MPO deficiency, a relatively common disorder, is associated with the absence of mature MPO but the presence of proMPO, consistent with a post-translational defect. Studies at the molecular level aimed at identifying the underlying genetic defect are thus far consistent with that hypothesis. In addition, the basis for the observed association between acquired MPO deficiency and some myeloid leukemias can now be studied at the molecular level using these probes.     

Inhibition of platelet aggregation by myeloid leukaemic cells demonstrated in vitro

The effect of myeloid leukaemic cells, cells of the promyelocytic cell line HL-60 and normal polymorphonuclear granulocytes (PMN), enclosed in dialysis tubes, on the aggregation of allogeneic normal platelets after induction with arachidonic acid (AA) and with adenosine diphosphate (ADP) was investigated in vitro. Inhibition of aggregation was seen after preincubation of the platelets with leukaemic blood or bone marrow cells from 7/14 patients belonging to various FAB groups and with HL-60 cells, but not with normal PMN (14/14 cases). A dose-dependent inhibition was seen after lysis of some leukaemic cells with anti-human rabbit antiserum and active complement. Lysis of normal PMN inhibited platelet aggregation slightly and inconstantly and only at higher cell concentrations. Platelet inhibitory activity was not related to a higher rate of metabolism of the leukaemic cells which inhibited platelet aggregation since they did not differ from the cells not inhibiting aggregation with respect to heat production. Neither was a non-specifically increased cell membrane permeability the cause of the release of platelet inhibitory factor(s) since the release of 51Cr-labelled leukaemic cells was not related to the inhibition of platelet aggregation.     

Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells.

The tumor antigen p53 is overproduced in transformed cells of various species, including man. HL-60 is an exceptional human tumor cell line that does not express this protein. Hybridization of polyadenylylated mRNA of these cells with a human p53 cDNA probe (p53-H14), which we cloned, had indicated a total absence of the mature-size (3.0 kilobases) or any aberrant p53 mRNA species. Analysis of the genomic HL-60 DNA indicated that the p53 gene in these cells was significantly altered. Most of the gene was deleted, and the residual p53 sequences of these cells, which show weak homology, mapped to the corresponding 5' region of the p53 gene. In agreement with previously documented results, we found that HL-60 cells have an amplified c-myc gene. We suggest that the deficiency of the p53 protein in HL-60 cells could have been overcome by using an alternative metabolic pathway. The c-myc product is a candidate for such an alternative protein.     

Expression of cytolytic functions in HL-60 leukaemic cells after induction of polymorphonuclear leukocyte differentiation

Summary Mature polymorphonuclear leukocytes (PMN) are capable of mediating phorbol myristate acetate (PMA)- and antibody (A)-dependent cellular cytotoxicity (DCC) against ox red blood cells (ORBC) by using oxidative means. The purpose of the present study was to investigate the acquirement of these cytotoxic functions during PMN ontogeny, using the promyelocytic HL-60 cell line as a model for PMN differentiation. HL-60 cells were induced to differentiate along the PMN pathway by exposure to dimethyl sulfoxide (DMSO). Uninduced HL-60 cells were found to be completely devoid of PMA-DCC and ADCC activity. DMSO-induced cells progressively acquired the capacity to kill ORBC and to undergo the activation of oxidative metabolic burst when triggered by PMA. Despite 40% of them also were capable of binding IgG-sensitized ORBC, no ADCC activity and respiratory burst activation was observed: this finding indicates that maturing HL-60 cells require a more complete maturation than that induced by DMSO to actually exert ADCC. Together the results suggest that: a. the acquirement of both PMA-DCC and ADCC potential is a post-promyelocytic event; b. the cytotoxicity activating stimuli, PMA and IgG-coated targets, follow different post-receptor transductional pathways to trigger the effector cell lytic systems: only the PMA receptor-linked pathway develops during DMSO-driven differentiation of HL-60 cells.Abbreviations ADCC antibody-dependent cellular cytotoxicity - DMSO dimethyl sulfoxide - EA_G ox red blood cells sensitized with IgG antibodies - EA_G-RFC EA_G rosette forming cells - MPO myeloperoxidase - NASDCA naphthol AS-D chloracetate esterase - NEM N-ethyl-maleimide - O ₂ ^– superoxide anion(s) - ORBC ox red blood cell(s) - PMA Phorbol myristate acetate - PMA-DCC PMA-dependent cellular cytotoxicity - PMN Polymorphonuclear leukocyte(s) - RPMI-FCS RPMI 1640 medium plus 10% heat-inactivated fetal calf serum     

Actin polymerization and its relationship to locomotion and chemokinetic response in maturing human promyelocytic leukemia cells

We studied actin polymerization in the HL-60 human promyelocytic leukemia cell line during induced myeloid maturation and its relationship to the rate of locomotion (ROL). The percent G-actin (of total actin) was measured by DNAase I inhibition, F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained cells, and ROL was measured by computer-assisted analysis of the tracks of individual cells. Uninduced HL-60 cells moved slowly (2.3 +/- 1.0 microns/min) and showed no change in ROL or in the state of actin polymerization when stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP). Nonstimulated cells induced to differentiate with dimethylformamide had no change in the degree of actin polymerization but exhibited a mean (m) ROL similar to normal human polymorphonuclear leukocytes (PMN) (8.6 +/- 1.4 micron/min [HL-60 cells] v 7.8 +/- 1.8 microns/min [PMN]. When induced HL-60 cells were stimulated with fMLP, actin polymerization occurred. The F-actin content increased, as determined by FACS analysis of NBD-phallacidin-stained cells, and the percentage of G-actin decreased, as determined by a 24.5% decrease in DNAase I inhibitory activity. However, induced HL-60 cells stimulated with fMLP did not increase their mROL. These studies show that, unlike normal human PMN, chemotactic peptides can cause an intracellular biochemical change that is not associated with a chemokinetic response in induced HL-60 cells. The HL-60 cell line may be a useful model to study the development of chemotactic peptide-mediated actin polymerization during myeloid cell maturation.     

Degradation of cartilage proteoglycans by myeloid leukemia cells

Polymorphonuclear leukocytes contain proteases that are capable of degrading articular cartilage matrix in disease states such as rheumatoid arthritis and osteoarthritis. In this study, the HL-60 human promyelocytic leukemia cell line was examined for ability to degrade cartilage proteoglycans. The HL-60 cells contained proteoglycan-degrading enzymes, which may contribute to the joint inflammation sometimes seen in acute leukemia. However, the protease activity was much less than in mature neutrophils and was not enhanced by the induction of myeloid maturation with dimethyl sulfoxide or retinoic acid. The diminished enzyme activity of induced HL-60 cells compared to normal neutrophils is another functional deficiency of these cells.     

The role of cellular maturation in neutrophil heterogeneity

Previous studies have shown that many neutrophil (PMN) characteristics are heterogeneous but the origin of PMN heterogeneity is unknown. It is unclear if PMN functional heterogeneity is secondary to maturational differences or due to distinct subpopulations of cells that possess different functional capacities. The PMN 31D8 antigen is a useful probe for evaluation of PMN subpopulations. The majority of PMNs (approximately 85%) exhibit a high intensity fluorescence after 31D8 monoclonal antibody (MoAb) labeling (31D8 enriched or "bright" PMNs) as determined by flow cytometric analysis. These cells are more functional than cells with low intensity fluorescence (31D8 diminished or "dull" PMNs). Various immunologic, clonogenic and functional techniques were used to study the expression of the 31D8 antigen in HL-60 cells and myeloid cells in order to evaluate antigenic and functional heterogeneity during morphologic maturation. The results of this study indicate that the percentage of 31D8 antigen positive (31D8 antigen enriched and diminished) bone marrow cells increases from 20 +/- 11% in myeloblast cells to 68 +/- 10% in promyelocytes, 93 +/- 2% in myelocytes and 99 +/- 1% in bands and PMNs. 31D8 antigen enriched cells first appear at the myelocyte stage (32 +/- 10%) and increase in bands (52 +/- 13%), marrow PMNs (62 +/- 13%) and peripheral blood PMNs (88 +/- 4%). These data indicate that the heterogeneous expression of 31D8 antigen in PMNs is due, at least in part, to maturational differences within the PMN population and raise the possibility that other heterogeneously expressed PMN characteristics are also maturationally derived. They also suggest that 31D8 antigenic expression may be a more precise indicator of myeloid functional maturation than maturation as identified by cellular morphology.     

Partial Myeloperoxidase Deficiency in a Case of Preleukaemia

A patient with a refractory anaemia preceding acute myeloblastic leukaemia had an increased susceptibility to infection due to Staphylococcus aureus. 36% of neutrophils lacked myeloperoxidase (MPO) activity and, in vitro, these polymorphonuclear neutrophils (PMN) had a defect of bactericidal activity against Staphylococcus aureus. Cytochemical studies of phagocytosis with the electron miscroscope have shown that the degranulation of primary granules (MPO+ or MPO-) was normal after phagocytosis of Escherichia coli which were normally lysed. A defective destruction of Staphylococcus aureus and Candida albicans was observed in some PMN with or without MPO activity, suggesting that MPO deficiency itself was not the only cause of this defect. In PMN which appeared normal, most MPO(+) granules were unable to fuse with the phagocytic vacuole containing intact germs even after 90 min of contact. There was, therefore, in addition to a partial MPO deficiency, a defect in cellular degranulation. This defect, the mechanism of which is unknown, may be in part responsible for the defective bacterial degradation.     

Differentiation stimuli induce receptors for plasma fibronectin on the human myelomonocytic cell line HL-60

Plasma fibronectin (Fn) induces phagocytosis of C3b-opsonized sheep erythrocytes (EC3b) by human peripheral blood monocytes. However, Fn does not induce erythrophagocytosis of EC3b by human polymorphonuclear leukocytes (PMN), unless the PMN have been exposed to C5a or N-formyl- methionyl-leucyl-phenylalanine. Because of this difference, it is of great interest to examine Fn binding to cells that possess the capacity to differentiate into either granulocytes or monocytes. Hence, we have examined the consequences of Fn binding to the human myelomonocytic cell line, HL-60, both before and after in vitro differentiation of the HL-60, along a monocytoid or a granulocytoid pathway. Fn receptors were not found on undifferentiated HL-60, but several differentiating agents promoted the HL-60 binding of Fn-coated microspheres (Fn-ms). The peak of Fn-ms binding occurred four to five days after the induction of differentiation with dimethylsulfoxide (DMSO), and two days after induction by PMA. In addition, cells that differentiated along either the monocytoid or the granulocytoid pathway showed a marked increase in the phagocytosis of both IgG-coated erythrocytes (EA) and EC3b when they were exposed to Fn. Comparison of the effects of anti-Fn monoclonals on the binding of Fn-ms to the monocytes, PMN, and HL-60 showed that the same monoclonals block Fn-ms-binding and Fn-induced EC3b phagocytosis by all three cell types. Two monoclonal antibodies, M1/70 and A6F10, directed against membrane antigens on PMN and monocytes, inhibited Fn-ms binding. Both also blocked Fn-induced EC3b ingestion by these cells. However, neither antibody blocked Fn-ms binding or EC3b ingestion by differentiated HL-60. We conclude that differentiated HL-60 cells express functionally active Fn receptors, similar to monocytes and activated PMN, which, nonetheless, differ from normal cells in their association with the antigens recognized by M1/70 and A6F10.