Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation

Differentiation and maturation of myeloid cells is characterized by the sequential acquisition of two distinct cytoplasmic granule subsets, azurophil granules and specific granules. We recently showed the existence of a third granule subset, gelatinase granules. To investigate whether appearance of gelatinase granules marks a further step in maturation of myeloid cells beyond the appearance of specific granules, we sorted normal human bone marrow cells into one of three groups according to maturity by centrifugation on Percoll density gradients. The biosynthesis of myeloperoxidase (MPO) (an azurophil granule marker), lactoferrin and neutrophil gelatinase-associated lipocalin NGAL (specific granules markers) and gelatinase was then studied in each of these groups. We found that gelatinase was synthesized mainly in the group containing band cells and segmented cells. This contrasted with lactoferrin and NGAL, which were synthesized almost exclusively in the group containing myelocytes and metamyelocytes, and with MPO, which was mainly synthesized in the group containing myeloblasts and promyelocytes. Immunocytochemistry was in full agreement with the biosynthesis data, and showed that gelatinase appears in band cells, whereas NGAL and lactoferrin both appear in myelocytes. Thus, acquisition of gelatinase granules marks a step in neutrophil differentiation beyond the appearance of specific granules.     

Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.

A human leukemic cell line (designated HL-60) has recently been established from the peripheral blood leukocytes of a patient with acute promyelocytic leukemia. This cell line displays distinct morphological and histochemical commitment towards myeloid differentiation. The cultured cells are predominantly promyelocytes, but the addition of dimethyl sulfoxide to the culture induces them to differentiate into myelocytes, metamyelocytes, and banded and segmented neutrophils. All 150 clones developed from the HL-60 culture show similar morphological differentiation in the presence of dimethyl sulfoxide. Unlike the morphologically immature promyelocytes, the dimethyl sulfoxide-induced mature cells exhibit functional maturity as exemplified by phagocytic activity. A number of other compounds previously shown to induce erythroid differentiation of mouse erythroleukemia (Friend) cells can induce analogous maturation of the myeloid HL-60 cells. The marked similarity in behavior of HL-60 cells and Friend cells in the presence of these inducing agents suggests that similar molecular mechanisms are involved in the induction of differentiation of these human myeloid and murine erythroid leukemic cells.     

Lactoferrin gene promoter: structural integrity and nonexpression in HL60 cells.

Lactoferrin is a member of the transferrin family of iron-binding proteins. It is found in several glandular epithelial tissues and human neutrophils, where it is localized to secondary granules. To examine the mechanisms controlling lactoferrin gene expression in neutrophils and defects in its expression in acute leukemia, we have cloned a lactoferrin cDNA from a chronic myelogenous leukemia library, and used it to obtain genomic clones representing the chromosomal lactoferrin gene. Using polymerase chain reaction, primer extension, and S1 analysis, we have identified the 5' end of the lactoferrin mRNA. We have defined a putative promoter region for the gene, and characterized its first two exons. In addition, we have examined the structure of these regions in DNA from HL60 cells. HL60 is a leukemic cell line that undergoes phenotypic neutrophil maturation on exposure to dimethyl sulfoxide (DMSO). However, the cells cannot be induced to express any secondary granule protein genes. We have shown that the 5' end of the lactoferrin gene, including the putative promoter region, is entirely normal in HL60. By Northern analysis, nuclear run-on studies, and primer extension assays we have shown that the gene is not transcribed in DMSO-induced HL60 cells. This supports the hypothesis that the defect in HL60 is an abnormality in the production or activity of a transacting regulator of lactoferrin gene expression.     

Targeting of proteins to granule subsets is determined by timing and not by sorting: The specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells.

The mechanism of protein targeting to individual granules in cells that contain different subsets of storage granules is poorly understood. The neutrophil contains two highly distinct major types of granules, the peroxidase positive (azurophil) granules and the peroxidase negative (specific and gelatinase) granules. We hypothesized that targeting of proteins to individual granule subsets may be determined by the stage of maturation of the cell, at which the granule proteins are synthesized, rather than by individual sorting information present in the proteins. This was tested by transfecting the cDNA of the specific granule protein, NGAL, which is normally synthesized in metamyelocytes, into the promyelocytic cell line HL-60, which is developmentally arrested at the stage of formation of azurophil granules, and thus does not contain specific and gelatinase granules. Controlled by a cytomegalovirus promoter, NGAL was constitutively expressed in transfected HL-60 cells. This resulted in the targeting of NGAL to azurophil granules as demonstrated by colocalization of NGAL with myeloperoxidase, visualized by immunoelectron microscopy. This shows that targeting of proteins into distinct granule subsets may be determined solely by the time of their biosynthesis and does not depend on individual sorting information present in the proteins.     

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 and Lactoferrin of Blood Neutrophils and Plasma in Chronic Granulocytic Leukaemia

Myeloperoxidase, restricted to primary granules, and lactoferrin, restricted to secondary granules, were determined in plasma and neutrophils of peripheral blood in chronic granulocytic leukaemia (CGL). Plasma myeloperoxidase was increased 2–3 times while plasma lactoferrin increased 2–8 times. This discrepancy indicates different modes of release or elimination. A correlation was found between the leucocyte count and plasma myeloperoxidase or lactoferrin. A correlation was also found between cellular and plasma levels of lactoferrin but not for myeloperoxidase indicating the source for plasma lactoferrin to be circulating leucocytes, which may not be the case for plasma myeloperoxidase. Decreased neutrophil lactoferrin was found in 71% of the CGL cases while myeloperoxidase was decreased in 18 %. Serial studies on individual CGL subjects showed low cellular lactoferrin during phases with rapidly expanding leucocytosis indicating defective maturation of neutrophils or abnormal release because of prolonged intravascular life-span.     

Myeloperoxidase and lactoferrin of blood neutrophils and plasma in chronic granulocytic leukaemia.

Myeloperoxidase, restricted to primary granules, and lactoferrin, restricted to secondary granules, were determined in plasma and neutrophils of peripheral blood in chronic granulocytic leukaemia (CGL). Plasma myeloperoxidase was increased 2-3 times while plasma lactoferrin increased 2-8 times. This discrepancy indicates different modes of release or elimination. A correlation was found between the leucocyte count and plasma myeloperoxidase or lactoferrin. A correlation was also found between cellular and plasma levels of lactoferrin but not for myeloperoxidase indicating the source for plasma lactoferrin to be circulating leucocytes, which may not be the case for plasma myeloperoxidase. Decreased neutrophil lactoferrin was found in 71% of the CGL cases while myeloperoxidase was decreased in 18%. Serial studies on individual CGL subjects showed low cellular lactoferrin during phases with rapidly expanding leucocytosis indicating defective maturation of neutrophils or abnormal release because of prolonged intravascular life-span.     

Ultrastructural, immunochemical, and cytochemical study of myeloperoxidase in myeloid leukemia HL-60 cells following treatment with succinylacetone, an inhibitor of heme biosynthesis.

Myeloperoxidase (MPO) is a heme-containing glycoprotein found in the primary granules (or azurophilic granules) of human polymorphonuclear leukocytes. In the present study, cultured myeloid leukemia HL-60 cells were exposed for 0-72 h to 250 microM 4,6-dioxoheptanoic acid (succinylacetone, SA), a specific inhibitor of heme biosynthesis, and the effects were evaluated using ultrastructural, immunochemical, and cytochemical methods. En bloc peroxidase staining of glutaraldehyde-fixed cells was accomplished with a 30-min exposure to 3,3'-diaminobenzidine (DAB) tetrahydrochloride. Ultrastructural examination revealed that peroxidase reactivity in the endoplasmic reticulum (ER) was relatively unchanged for 8 h and decreased between 12 and 24 h; however, ER lacked DAB-reactive peroxidase at 48-72 h. Peroxidase reactivity in the ER reappeared within 4 h after removal of SA. Seventy-two hours after exposure to SA the number of condensed cytoplasmic granules stained with DAB was significantly decreased, and many of the granules had a "target" appearance with a central DAB-reactive dense core. Staining of mitochondria was observed with overnight exposure to DAB and persisted in HL-60 cells treated 72 h with SA. Mitochondrial and nuclear morphology appeared unaltered. Immunostaining of MPO in thin sections of paraformaldehyde/glutaraldehyde-fixed unosmicated HL-60 cells, embedded in Lowicryl K4M, was accomplished with sequential exposure to an affinity-purified monospecific rabbit antibody to HL-60-MPO and protein A conjugated to 5- or 10-nm colloidal gold. Compared to untreated control HL-60 cells, cells exposed to SA for 48 h exhibited comparable to increased immunoreactive MPO in the ER, despite the absence of heme-dependent peroxidase reactivity. The data indicate that SA inhibits formation of enzymatically active MPO and that in the presence of SA, the ER contains a form(s) of MPO that lacks enzymatic reactivity.     

Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukemia cells (HL60) treated with chemical inducers

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O- tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.     

Cellular and subcellular localization of the bactericidal/permeability- increasing protein of neutrophils

Human and rabbit polymorphonuclear leukocytes contain a bactericidal/permeability-increasing protein (BPI), a potent cytotoxin active specifically against gram-negative bacteria. To identify the cell population(s) producing BPI, we have examined mature and immature human blood cells for BPI by immunofluorescence of intact cells and radioimmunoassay and bioassay of cell extracts. By immunofluorescence and radioimmunoassay of cells from peripheral blood, BPI was detected only in neutrophils; immunofluorescent staining was punctate, indicative of the granule localization of BPI. Nearly all (greater than 90%) BPI was recovered during the subcellular fractionation of neutrophils (N2 cavitation and discontinuous Percoll gradient) in fractions containing primary granules. Little BPI was released from intact cells during degranulation (cytochalasin B and f-Met-Leu-Phe) or could be extracted from isolated granules with salt or weak acid, which suggests that most granule-associated BPI is membrane bound. Double staining of bone marrow smears for BPI and lactoferrin revealed BPI only in neutrophil precursors including (pro)myelocytelike cells lacking lactoferrin, a marker of neutrophil secondary granules. Of several human cell lines tested, only the promyelocytelike HL-60 (and to a lesser extent, KG-1) cells contained BPI. BPI was present in a more mature subpopulation (less than 25%) of untreated HL-60 cells, recognized by surface marker analysis (rosetting with IgG-sensitized sheep RBC, the absence of proliferation-associated cell surface antigen). Induction of neutrophilic or monocytic differentiation caused, respectively, a small (approximately 50%) rise or fall in the BPI content. These findings indicate that BPI is a specific product of the neutrophil lineage and, hence, of the specialized cytotoxic apparatus of the neutrophil that plays an essential role in host defense v gram-negative bacteria.     

Granulocytopoiesis in Human Bone Marrow Cultures Studied by Means of Kinematography

1. In short-term human bone marrow cultures, the generation times ofthree myelocytes were measured by phase contrast kinematography to be29-30.5 hours. Two of these myelocyte generations were in direct successionof each other.

2. The mean mitotic time of 93 myelocytic mitoses was found to be 88 ±20 minutes normally and 142 ± 13 minutes in 13 leukemic blast cells. Thisdifference was statistically significant.

3. The maturation of myelocytic cells after their last mitosis from themetamyelocyte into mature granulocytes was followed and found to varybetween 7 and 37 hours. This time difference was the result of the fact thatone of the cells underwent a further division, where the three sister cells didnot divide but matured only. All five final cells acquired the characteristics ofband forms at about the same time. In eight other cells, the metamyelocyte-bandform transition time was found to be 7 to 30 hours. In all cells studied,the morphologic characteristics changed repeatedly between those consideredtypical for myelocytes and metamyelocytes. Thus, metamyelocytes werefound to undergo division under the conditions of this study.

4. No evidence was found for amitotic divisions. However, the development of tetraploid cells was observed to be the result of endomitosis, ofpostmitotic formation of binucleated cells, or of formation of giant metamyelocytes by nuclear fusion of a binucleated cell.

Submitted on August 20, 1964 Accepted on January 13, 1965     

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.     

An Investigation into the Development and Fate of Neutrophil Giant Metamyelocytes using the Techniques of Electron Microscopy and High Resolution Autoradiography

The neutrophil giant metamyelocytes present in vitamin B₁₂- and folate-deficient patients were studied using the techniques of electron microscopy and electron microscope autoradiography. The ultrastructural features of the cytoplasm of a proportion of these cells resembled those of promyelocytes and myelocytes, particularly with respect to the types of neutrophil granules present. This finding suggests that the giant metamyelocytes result from an abnormal type of development in promyelocytes and myelocytes which have been arrested or retarded in their progress through the cell cycle. The hypothesis that giant metamyelocytes eventually die within the marrow was supported by the observations that a significant proportion of these cells contain intracytoplasmic autophagic vacuoles, that some giant metamyelocytes suffer from a marked depression of RNA and protein synthesis and that degenerating giant metamyelocytes can be recognized within the cytoplasm of some bone marrow macrophages.     

Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents.

Human promyelocytic leukemia cells (HL-60) were induced to differentiate into mature cells by the tumor-promoting agent phorbol-12-myristate-13-acetate and other related phorbol diesters. Differentiation was determined by an increase in the percent of myelocytes, metamyelocytes, and other mature myeloid cells as well as by an increase in the percent of phagocytizing cells. Induction of differentiation could be determined after 2 days of treatment with phorbol-12-myristate-13-acetate at a dose as low as 6 X 10(11) M. A correlation was found between reported tumor-promoting activity of a series of phorbol esters and their ability to induce myeloid differentiation and to inhibit cell growth. It is suggested that tumor-promoting agents like chemicals that induce terminal differentiation in these cells, at extremely low concentrations, may be used as a tool in the study of the control of cell growth, cell differentiation, and malignancy in human leukemic cells.     

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.     

A Quantitative Assessment of Neutrophil Marrow in Seven Patients with Chronic Granulocytic Leukaemia

Neutrophil marrow cellularity was determined in seven patients with chronic granulocytic leukaemia (CGL). The size of the mitotic pool (promyelocytes and myelocytes) and the number of metamyelocytes and bands and of segmented neutrophils in the postmitotic pool were determined from measurements of neutrophil--erythroid ratios in marrow biopsy sections and ferrokinetic estimates of marrow normoblasts. A section mitotic index was calculated in each patient from the numbers of mitotic figures and mitotic pool cells counted on marrow sections. Basal values previously established in normal subjects for the mitotic pool, for metamyelocytes and bands, and for segmented neutrophils, were 2.11 +/- 0.36 x 10(9) cells/kg, 3.33 +/- 0.61 x 10(9) cells/kg, and 2.26 +/- 0.42 x 10(9) cells/kg, respectively (+/- 1 SD, n = 13). The basal section mitotic index was 0.07 +/- 0.01 (+/- 1 SD, n = 13). In the seven patients with CGL the mitotic pool comprised 3.71--25.70 x 10(9) cells/kg, metamyelocytes and bands 7.70--51.02 x 10(9) cells/kg, and segmented neutrophils 3.45--28.81 x 10(9) cells/kg. Mitotic indices ranged from 0.04 to 0.10. No relationship was found between marrow cellularity and blood neutrophil count. A negative correlation existed between mitotic pool cellularity and mitotic index (r = -0.76, n = 7 pairs). The results provide quantitative affirmation of neutrophil marrow hyperplasia and of increased neutrophil production by the marrow in CGL.