Expression of two natural killer cell antigens, H-25 and H-366, by human immature myeloid cells and by erythroid and granulocytic/monocytic colony-forming units

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony- forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.     

Characterization of monoclonal antibodies to human monocytes

A set of monoclonal antibodies reacting with human peripheral blood monocytes is characterized. Two of these antibodies also bind to a fraction of granulocytes (BL-M/G-1) respectively to all of them (BL-M/G-2). On the other hand the BL-M-3 appears to be specific for monocytes only. All three monoclonals do not react with enriched B and T lymphocytes, platelets, erythrocytes and several lymphoid or erythroid permanent cell lines. The antigens recognized by all three monoclonals are expressed by monoblastic cell line U-937, whereas the myeloid line HL-60 and the cell line KG-1 express only antigen recognized by monoclonal antibody BL-M/G-2.     

Analysis of antigenic determinants on human monocytes and macrophages

Mo1, 2, 3, and 4, and Plt-1 are a series of five distinct antigens detected on the surface of human peripheral blood monocytes by mouse monoclonal antibodies. Mo2 and 3 are restricted to the monocyte- macrophage series, while Mo1, as previously reported, is also expressed by human granulocytes and null cells. Mo3, as distinguished from Mo1 and Mo2, is weakly expressed by virgin peripheral blood monocytes but becomes well expressed if monocytes are cultured overnight at 37 degrees C. Mo4 is coexpressed by monocytes and platelets, while Plt-1 appears to be a platelet-specific antigen whose detection on monocytes reflects adherence of platelets to monocyte membranes. That Mo2-4 are true monocyte antigens is demonstrated by their resynthesis following protease treatment of monocytes (Mol expression is resistant to proteolytic digestion). During myeloid-monocyte differentiation, the Mo antigens are infrequently expressed by immature myeloid cells but are found at higher frequency on leukemic monocytic forms. Macrophages from cultured peripheral blood monocytes and HL-60 cells exposed to lymphokines or phorbol diester express Mo1-4, but noncirculating peritoneal macrophages lack Mo3. The Mo antigens are differentiation markers whose expression reflects membrane heterogeneity during myeloid- monocyte-macrophage maturation.     

Somatic cell hybrid analyses of hematopoietic differentiation

A differentiated cell expresses an entire set of specialized features. Somatic cell hybridization provides a method to examine control of gene regulation. We studied the expression of tissue-specific features in hybrids between human promyelocytes (HL-60) and human Burkitt's lymphoma cells (P3HR-1). Two hybrid lines, HP-1 and HP-2, and 18 hybrid clones were established and confirmed by karyotype, isozyme, and surface antigen analyses. The hybrids extinguished the 10 myeloid (HL- 60) features that we examined including myeloid morphology, histochemistry, and functions that included response to colony- stimulating factor and ability to differentiate to granulocytes or macrophages. In contrast, the hybrids synthesized immunoglobulin and expressed Epstein-Barr nuclear, early, and viral capsid antigens similar to the P3HR-1 lymphoid parental line. Results are contrasted to the findings when P3HR-1 lymphocytes are fused to human erythroid- myeloid cells (K562). Taken together, our results suggest that phenotypic differences between human myeloid and lymphoid cells in the hematopoietic lineage involve mutually exclusive programs and may possibly be mediated by the activity of diffusible, transacting molecules.     

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.     

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.     

Specificity and diagnostic implications of the reactivity pattern of a panel of monoclonal antibodies against myeloid leukemia cells

The immunological phenotypes of leukemia cell samples from 60 patients, of whom 54 had acute myeloid leukemia (AML), were assessed with a panel of monoclonal antibodies (Mabs) with specificity for the following epitopes: Epitopes associated with myeloid leukemia cells, Epitopes expressed only on immature myeloid cells (or subsets) and on monocytes, Epitopes only expressed on granulocytes or on granulocytes and mature myeloid cells (promyelocytes, myelocytes and monocytes), Epitopes on HLA-class II (DR) and HLA-class I molecules and on insulin receptors. This panel of Mabs proved useful to identify leukemia cells in blood and to assess their myeloid origin. The panel of Mabs was found also to be useful for immunophenotyping of leukemia cells. Furthermore, the analysis revealed considerable variations in the immunological phenotype of AML cells, reflecting antigenic heterogeneity within the individual leukemia cell population as well as abnormal or no expression of histocompatibility antigens and insulin receptors in some samples. Some of the Mabs bound preferentially to subgroups in the French-American-British (FAB) classification.     

Pattern of expression of HLA-DR and HLA-DQ antigens and mRNA in myeloid differentiation

We examined the expression of HLA-DR and HLA-DQ antigens and mRNA from myeloid and lymphoid cells obtained from normal volunteers and established cell lines. Cytofluorometric analysis and immunoprecipitation were performed using murine monoclonal antibodies specific for HLA-DR (L-243) and HLA-DQ (Leu 10). The expression of mRNA for HLA-DR and HLA-DQ chains was determined by Northern blot and RNA dot-blot analysis. Lymphoid cell lines expressed both HLA-DR and HLA-DQ antigens, with consistently higher levels of expression of DR. Myeloid cell lines of early myeloblast or bipotent (myeloid-erythroid) phenotype (KG-1, KG-1a, HEL) expressed HLA-DR at high levels, whereas cell lines manifesting a greater degree of myeloid maturation (ML-3, HL- 60, U937) expressed DR at low or undetectable levels. The HLA-DQ antigen was expressed at low levels on the surface of KG-1 and KG-1a cells but was not detectable on other myeloid cell lines. The expression of mRNA for HLA-DR and HLA-DQ chains paralleled the pattern of expression of the respective antigens. The HL-60 and U-937 cells stimulated to differentiate in vitro to macrophages with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] were induced to express detectable levels of HLA-DR antigens. Exposure to gamma-interferon (gamma-IFN) increased the expression of HLA-DR antigens by all myeloid cell lines. Induction of differentiation in vitro with either 1,25(OH)2D3 or dimethyl sulfoxide potentiated this effect of gamma-IFN. Expression of the HLA-DQ antigens was increased on KG-1 myeloblasts after exposure to gamma-IFN. HLA-DQ expression could not be detected on other myeloid cell lines after exposure to gamma-IFN, nor was HLA-DQ expression stimulated by gamma-IFN after HL-60 and U-937 cells were induced to differentiate to macrophagelike cells in vitro. These results provide additional evidence that expression of the HLA-DR and HLA-DQ genes may be independently regulated in human myeloid cells.     

The expression and modulation of human myeloid-specific antigens during differentiation of the HL-60 cell line

Antigenic changes detected by myeloid-specific monoclonal antibodies on HL-60 cells induced to differentiate by various chemical mediators were investigated using flow cytometry. Antigen levels detected by monocyte- granulocyte-specific monoclonal antibodies AML-2-23, 61D3, and 63D3 increased dramatically after differentiation of HL-60 cells along the granulocytic pathway by the addition of dimethyl formamide (DMF), dimethylsulfoxide (DMSO), or cis-retinoic acid. The expression of these same antigens also increased in conjunction with monocytoid differentiation when HL-60 cells were treated with supernatants from leukocytes stimulated with phytohemagglutinin (PHA-LCM) or with mixed lymphocyte conditioned medium (MLC). In contrast, treatment of HL-60 cells with phorbol 12-myristate 13-acetate (PMA), which also induced differentiation along the monocyte pathway, had no effect on the expression of these monocyte-associated antigens. The expression of antigens on HL-60 cells recognized by the granulocyte-specified monoclonal antibodies PMN 6 and PMN 29 decreased after treatment of HL- 60 cells with PMA, but remained constant after treatment with DMF, DMSO, cis-retinoic acid, PHA-LCM, or MLC. These results suggest that normal myeloid differentiation may be dependent on various signals and that morphological and cell surface marker maturity may, under some conditions, be separable. The utility of the HL-60 cell line as a model of myeloid differentiation and for evaluation of inductive signals is discussed.     

bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.

The present study provides immunobiochemical and molecular data on the differentiation-linked expression of the bcl-2 proto-oncogene in normal and neoplastic myeloid cells. Using a recently developed monoclonal antibody (MoAb) to the bcl-2 molecule, staining of normal bone marrow myeloblasts, promyelocytes, and myelocytes, but neither monocytes nor most polymorphonuclear cells, was demonstrated. By two-color flow cytometric analysis, bcl-2 was evidenced in CD33+ and CD33+/CD34+ myeloid cells as well as in the more primitive CD33-/CD34+ population. The leukemic cell lines HL-60, KG1, GM-1, and K562 were bcl-2 positive together with 11 of 14 acute myeloid leukemias (AML) and three of three chronic myeloid leukemias (CML) in blast crises; six of seven CML were negative. Among myelodysplastic cases, augmentation of the bcl-2 positive myeloblastic compartment was found in refractory anemia with excess of blasts (RAEB) and in transformation (RAEB-t). Western blots of myeloid leukemias and control lymphocytes extracts evidenced an anti-bcl-2 immunoreactive band of the expected size (26 Kd). Moreover, the HL-60 and KG1 cell lines, both positive for the bcl-2 protein, exhibited the appropriate size bcl-2 mRNA (7.5 Kb). These findings clearly indicate that the bcl-2 gene is operative in myeloid cells and that the anti-bcl-2 MoAb identifies its product and not a cross-reactive epitope. Induction of HL-60 differentiation toward the monocytic and granulocytic pathways was accompanied by a marked decrease in bcl-2 mRNA and protein levels; bivariate flow cytometric analysis showed that the fraction becoming bcl-2 negative was in the G1 phase of the cell cycle. These data establish that the bcl-2 proto-oncogene is expressed on myeloid cells and their progenitors and is regulated in a differentiation-linked manner.     

Leukemia cell lines can replace monocytes for mitogen-induced T-lymphocyte responses: this accessory function is dependent upon their differentiation stage.

Highly purified peripheral T lymphocytes do not proliferate in response to phytohemagglutinin A or concanavalin A, unless adherent HLA-DR+ monocytes are added as accessory cells. The accessory function (AF) of monocytes is mediated through the release of interleukin-1 (IL-1). We here report that cells from three human leukemic lines--K562, HL-60, and U-937--could exert AF and efficiently replace monocytes in a 72-hr mitogen-stimulated proliferation assay. This AF was clearly related to precise maturational stages of these cells, since the hematopoietic precursor K562 cells spontaneously exerted high AF but lost this property when treated with differentiation inducers such as sodium butyrate or phorbol 12-myristate 13-acetate (PMA). On the other hand, untreated HL-60 and U-937 cells exhibited no spontaneous AF, but they acquired this function when induced to differentiate either along the granulocytic pathway (dimethyl sulfoxide-treated HL-60 cells) or along the monocytic pathway (PMA-treated HL-60 and U-937 cells). Supernatants from PMA-triggered K562 or HL-60 cells allowed the proliferative response of murine thymocytes to phytohemagglutinin A and were therefore shown to contain IL-1. Analysis of phenotypical markers showed that AF and IL-1 production were not restricted to cells of the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL-60 cells. Thus, the expression of HLA-DR antigens is not required for AF and IL-1 production in response to mitogens. Human leukemia cell lines could provide useful sources of human IL-1.     

Down-regulation of human protein kinase C alpha is associated with terminal neutrophil differentiation.

We have established an RNase protection method to quantify the expression of mRNA for the human protein kinase C (PK-C) isoforms alpha, beta 1, beta 2, and gamma. This was used to investigate whether each isoform is differentially expressed during the differentiation of hematopoietic cells. Myeloid and lymphoid cells express PK-C alpha, beta 1, and beta 2 mRNAs in various proportions. PK-C gamma mRNA was detected in human brain, but not in hematopoietic cells. PK-C alpha mRNA decreases as HL-60 cells mature to a neutrophil phenotype in response to retinoic acid, but its abundance does not change during monocytic differentiation in response to vitamin D3. PK-C alpha mRNA and protein were undetectable in peripheral blood neutrophils, but are present in monocytes. The mRNAs for PK-C beta 1 and beta 2 isoforms increase during HL-60 differentiation and are expressed in both neutrophils and monocytes. Therefore, the PK-C alpha isoform is specifically down-regulated during human neutrophil terminal differentiation. These data suggest that mature neutrophil functions do not require the PK-C alpha isoform.     

Implication of expression of GDNF/Ret signalling components in differentiation of bone marrow haemopoietic cells

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) mediate their actions through a unique multicomponent receptor system composed of Ret receptor tyrosine kinase and glycosyl-phosphatidylinositol-linked cell surface proteins (designated GFRalpha-1 and GFRalpha-2). In the present study, expression of these signalling components in the process of differentiation of haemopoietic cells was investigated. Ret was expressed at variable levels in normal and malignant cells of the myelomonocyte lineage. Immunohistochemical analysis of human and mouse tissues revealed that Ret expression was increased in intermediate mature myeloid cells such as promyelocytes and myelocytes and decreased in mature granulocytes and monocytes. Consistent with this observation, when THP-1 monocytic and HL-60 promyelocytic leukaemia cells expressing Ret were differentiated toward macrophages or granulocytes by treatment of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans retinoic acid (RA), Ret expression strikingly decreased during differentiation. Expression of GDNF, NTN, GFRalpha-1 and GFRalpha-2 was undetectable in THP-1 and HL-60 cells as well as in bone marrow haemopoietic cells. In contrast, bone marrow stromal cells appeared to express GDNF, GFRalpha-1 and GFRalpha-2 but not Ret. These findings suggested that the interaction between stromal cells and Ret-expressing haemopoietic cells in the bone marrow microenvironment may play a role in the differentiation of myelomonocyte-lineage cells through activation of the GDNF/Ret signalling pathway.     

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.     

Expression of CD11, CDw15, and transferrin receptor antigens on human hematopoietic progenitor cells

The expression of transferrin receptor-associated antigens and of CD11 and CDw15 antigens was investigated on myeloid committed progenitor cells (CFU-GM day 10, CFU-GM day 7, and cluster-forming cells [CFC] day 4), on erythroid committed progenitor cells (BFU-E and CFU-E), and on multilineage progenitor cells (CFU-GEMM). Both complement-dependent cytotoxicity and fluorescence-activated cell-sorting assays were performed. Complement-dependent cytotoxicity appeared to be the more sensitive assay. Transferrin receptor-associated antigens appeared to be clearly present on all myeloid and erythroid committed progenitor cells, but were found to be only weakly expressed on CFU-GEMM. CD11 antigens appeared to be strongly expressed only on mature granulocytes, monocytes, and certain lymphocytes, but not significantly on myeloid committed precursor cells. Surprisingly, CD11 antigens were weakly, but significantly, present on CFU-E. CDw15 antigens appeared to be restricted to myeloid differentiation and were increasingly expressed from CFU-GM day 10 to CFC day 4. Thus, antitransferrin receptor, CD11, and CDw15 antibodies can be used to separate hematopoietic progenitor cells and may be useful tools in the study of hematopoietic differentiation.     

Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies

Within the hematopoietic system, monoclonal antibodies reactive with antigenic determinants, expressed in a lineage- and stage-restricted fashion, can be used to map myeloid differentiation. We have generated a series of monoclonal antibodies that reacts with myeloid-associated determinants on committed myeloid stem cells and their progeny. Their reactivity with peripheral blood cells was identified by immunofluorescence assays, with bone marrow cells by fluorescence- activated cell sorting, and with committed hematopoietic progenitor cells by both cytotoxic assays and fluorescence-activated cell sorting. Antibody 1G10, which has previously been reported to react with cells of the granulocytic lineage and with a minor subset of mature monocytes, was shown to react with granulocyte-macrophage colony- forming units (CFU-GM). Three antibodies not previously characterized (T5A7, L4F3, L1B2) were shown to react with both granulocytic and monocytic cells and in fluorescence-activated cell sorting studies to detectably stain granulocytic cells at different stages of maturation. These three antibodies also react with CFU-GM, two (L4F3 and L1B2) reacting with all CFU-GM, while T5A7 reacts with only a portion of the day 7 CFU-GM. Antibody L4F3 also reacts with a portion of erythroid burst-forming units (BFU-E). In contrast, the previously reported antibody 5F1, which reacts with monocytic cells, nucleated erythroid cells, and platelets, was shown to react with erythroid colony-forming units (CFU-E). Potential applications of these antibodies to studies of normal and malignant hematopoiesis are discussed.     

Changes in surface antigens of HL-60 cells during differentiation in vitro

Summary We have examined the pattern of binding of monoclonal antibodies OKOM 1, FMC 10, FMC 12, FMC 13, FMC 17 and FMC 33 to human promyelocytic leukaemia (HL-60) cells. We found that the expression of antigens detectable with FMC 17 and FMC 33 (specific for monocytes and macrophages) was increased by exposure of HL-60 cells to 1,25-dihydroxyvitamin D₃ but not by exposure of HL-60 cells to 12-tetradecanoyl phorbol-13-acetate (TPA). The antigen detected with the OKM 1 antibody was highly induced by TPA. The expression of granulocyte-specific antigens detected by FMC 10 and FMC 13 was increased during induction of granulocytic maturation; these antigens were retained during monocytemacrophage differentiation of HL-60 cells. We conclude that in some cases the expression of particular antigens during maturation of malignant cells proceeds normally while in other cases antigenic differences between leukaemic and normal cells at equivalent levels of maturation can be detected.     

The expression of myeloid-specific antigens on myeloid leukemia cells: correlations with leukemia subclasses and implications for normal myeloid differentiation

The expression of three distinct myeloid-specific cell surface antigens detected by monoclonal antibodies (PMN 6, PMN 29, and AML-2-23) on acute and chronic myeloid leukemia cells is correlated with blast cell morphology and normal myeloid cell antigen display. In studies on normal peripheral blood cells, monoclonal antibodies PMN 6 and PMN 29 have previously been shown to react exclusively with neutrophils while AML-2-23 reacts with both neutrophils and monocytes. The present report demonstrates that these antigens are absent from blast cells of patients with acute myelocytic leukemia (AML) classified as M1 and M2 in the French-American-British system and chronic myelocytic leukemia in myeloid blast crisis. However, leukemia cells with myelomonocytic morphology (M4) expressed all three antigens, while cells with pure monocytic features (M5) were generally only positive for AML-2-23. Based on the absence of these antigens on both leukemic and normal myeloblasts and granulocyte-monocyte progenitors and their characteristic patterns of display on more differentiated leukemic and normal cells, we propose a modified concept of normal myelopoiesis. In this hypothesis, the myeloblast is an uncommitted cell that gives rise to a series of intermediate precursors that acquire committment to either the granulocytic or monocytic lineage marked by the acquisition of specific cell surface markers.