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M W Fanger

Publications and source records attributed to M W Fanger.

At least 91 records · Page 5Linked to original sources
PubMed1985

Isolation and characterization of My23, a myeloid cell-derived antigen reactive with the monoclonal antibody AML-2-23.

In this study, we describe the isolation and characterization of My23, a human myeloid antigen defined by the monoclonal antibody (MoAb) AML-2-23. Cells of the HL-60 human promyelocytic cell line, when cultured in the presence of 1,25-dihydroxyvitamin D3 (calcitriol), express a surface protein of approximately 50 to 55 kilodaltons (Kd) which was immunoprecipitated with the AML-2-23 MoAb. Furthermore, after 2 days of exposure to calcitriol, HL-60 cells began to release My23 into culture medium, as determined by the ability of culture supernatant from these cells to block the binding of AML-2-23 to myeloid cells. My23 release was almost totally inhibited by incubation of cells at 4 degrees C, and was partially blocked by treatment of cells with cycloheximide or tunicamycin. The culture supernatant blocking factor, soluble My23, was identified as a 45 to 50 Kd protein by Western blot/immune overlay, using AML-2-23 and an 125I-labeled second antibody. My23, which was affinity-purified from culture supernatant, retained the ability to block AML-2-23 binding to myeloid cells. The affinity-purified antigen migrated on SDS-PAGE as a diffuse band in the m.w. range of 44 to 52 Kd. On treatment with endoglycosidase, the apparent m.w. of My23 decreased to approximately 40,000, indicating the presence of carbohydrate residues on My23. Serum from mice immunized with the purified antigen reacted with the same spectrum of myeloid cells as AML-2-23 MoAb, reacted with the My23 soluble protein in immunoblots, and competed with AML-2-23 for binding to myeloid cells. Binding of this antiserum to myeloid cells was blocked by cell supernatant from both monocytes and calcitriol-treated HL-60 cells, suggesting, along with results from m.w. determinations of the two preparations, that the soluble and cell surface forms of My23 are similar. Moreover, based on our finding that human plasma specifically inhibits the binding of AML-2-23 to myeloid cells, My23 may also be released in vivo. The enhanced expression of My23 on activated and more mature myeloid cells and its shedding or secretion by these cells is consistent with a functional role for My23.

Antibodies, Monoclonal↗
PubMed1984

Monoclonal antibodies PMN 6, PMN 29, and PM-81 bind differently to glycolipids containing a sugar sequence occurring in lacto-N-fucopentaose III.

Three monoclonal antibodies, PMN 6, PMN 29, and PM-81, bind myeloid cells. Antibodies PMN 6 and PMN 29 bind specifically to granulocytes but differ in their ability to bind some other cell lines [E. D. Ball, R. F. Graziano, L. Shen, and M. W. Fanger (1982) Proc. Natl. Acad. Sci. USA 79, 5374-5378]. Antibody PM-81, in addition to granulocytes, also binds to eosinophils, monocytes, and most acute myelocytic leukemia cells [E. D. Ball, R. F. Graziano, and M. W. Fanger (1983) J. Immunol. 130, 2937-2941]. Despite these differences, the binding of all three antibodies to cells was inhibited by the oligosaccharide, lacto-N-fucopentaose III [Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc]. Solid-phase radioimmunoassays using purified glycolipids containing sugar sequences found in lacto-N-fucopentaose III demonstrated different binding characteristics for each antibody. PM-81 bound lower concentrations of glycolipids than PMN 29, while PMN 6 required the highest concentration of glycolipids for binding. Autoradiography of thin-layer chromatograms of glycolipid antigens supported these results. The binding of these monoclonal antibodies to cells probably depends on the density of antigens on the cell surface, each antibody requiring a different density. Thus, cells containing antigen below a certain threshold concentration may not bind low-affinity antibodies.

Antibodies, Monoclonal↗
PubMed1984

Direct stimulation of ADCC by cloned gamma interferon is not ablated by glucocorticoids: studies using a human monocyte-like cell line (U-937).

We have used the macrophage-like cell line U-937 to demonstrate that recombinant gamma (immune) interferon (gamma-IFN) acts directly on the mononuclear phagocyte in the absence of other cell types to increase Fc receptor sites and antibody-dependent cellular cytotoxicity (ADCC). Incubation of U-937 for 18 hr with 2% gamma-IFN-rich supernatant, or with 10 U/ml of pure recombinant gamma-IFN, resulted in a seven-fold increase in Fc receptors as measured by the binding of radiolabeled IgG or fluoresceinated IgG and cytofluorography. Simultaneous measurement of ADCC for chick erythrocytes showed a seven-fold increase. This augmentation of Fc receptors and function was not ablated by an immunosuppressive cocn of the glucocorticoid dexamethasone. The potent effects of gamma-IFN both on surface receptors and effector functions of macrophages suggest that it is an important mediator in the efferent limb of immunity. Moreover, our findings that physiologic levels of glucocorticoids do not block activation of the mononuclear phagocyte support our view that glucocorticoids are immunosuppressive as a result of their action on gamma-IFN-producing cells. This would seem an important consideration in the development of potential strategies for obviating steroid-induced immunosuppression.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Gamma interferon induces monocytoid differentiation in the HL-60 cell line.

We investigated the ability of purified, recombinant DNA-derived interferons (IFN) to induce phenotypic changes in cells of the HL-60 promyelocytic leukemia cell line. Changes in cell surface markers detected by monoclonal antibodies as well as morphologic, histochemical, and functional changes were monitored. We found that gamma-IFN, but not alpha- or beta-IFN, induced the expression of antigens characteristic of monocytes and granulocytes (AML-2-23, 63D3, and 61D3), as well as changes in morphology consistent with monocytoid differentiation. These included induction of alpha-naphthyl acetate esterase, increased cell size, and a decrease in azurophilic granules. The gamma-IFN dose dependency and time course of the effect on antigen expression suggest that de novo protein synthesis was induced by gamma-IFN. The activity of gamma-IFN and of mixed-lymphocyte culture supernatant was blocked by a monoclonal antibody to gamma-IFN. Significant augmentation in the ability of the HL-60 cells to mediate antibody-dependent cellular cytotoxicity was induced by gamma-IFN. These findings suggest that gamma-IFN plays a role in the regulation of hematopoiesis.

Antibodies, Monoclonal↗
PubMed1984

Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol).

Recent studies have suggested that vitamin D may have other important biologic activities in addition to its well-characterized role in the maintenance of calcium homeostasis. Discovery of cytosolic receptors for vitamin D in human peripheral blood monocytes and lectin-stimulated lymphocytes prompted us to study the effects of 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, upon phytohemagglutinin (PHA)-induced lymphocyte blast transformation. We have found that calcitriol is a potent inhibitor of PHA-induced lymphocyte proliferation, achieving 70% inhibition of tritiated thymidine incorporation after 72 h in culture. Furthermore, calcitriol suppressed interleukin-2 (IL-2) production by PHA-stimulated peripheral blood mononuclear cells in a concentration-dependent fashion. Lastly, the suppressive effect of calcitriol on cellular proliferation was partially reversed by the addition of saturating amounts of purified IL-2. We conclude that calcitriol is a potent inhibitor of PHA-induced lymphocyte blast transformation and that this effect is mediated, in part, through suppression of IL-2 production. Thus, calcitriol appears to possess immunoregulatory properties that have been unappreciated heretofore.

Calcifediol↗
PubMed1984

Monoclonal antibodies reactive with small cell carcinoma of the lung.

Murine monoclonal antibodies (MoAb) reactive with antigens associated with small cell carcinoma of the lung (SCCL) were prepared and partially characterized. Four were selected for further study on the basis of their lack of reactivity with normal leukocytes and erythrocytes. These MoAb, designated SCCL-41, SCCL-114, SCCL-124, and SCCL-175, are all IgM immunoglobulins. The binding of these MoAb to patient-derived SCCL tumor cells, SCCL cell lines, and non-SCCL cell lines was studied by indirect immunofluorescence and flow cytometry. Considerable heterogeneity in the expression of these cell surface antigens was noted among both the patient-derived tumor cells and the SCCL cell lines. One of the MoAb, SCCL-175, reacted with 7 of 7 patient-derived tumor cell samples and 9 of 10 SCCL cell lines. None of the antigens defined by these MoAb were expressed on non-SCCL lung tumor cell lines. SCCL-175 reacted with cells from both a choriocarcinoma and a colon carcinoma cell line, whereas the other 3 MoAb were unreactive with these and several other tumor cell lines. These MoAb may be useful in the diagnosis and subclassification of SCCL tumors.

Animals↗
PubMed1984

Modulation of class I HLA antigens on HL-60 promyelocytic leukemia cells by serum-free medium: re-induction by gamma-IFN and 1,25-dihydroxyvitamin D3 (calcitriol).

During studies on the effect of different nutrient media on the growth and differentiation of the HL-60 promyelocytic leukemia cell line, we found that the density of the class I HLA antigens is profoundly decreased on cells cultured in a serum-free medium. The ability of recombinant DNA-derived interferons (IFN) and a number of myeloid differentiating agents to induce re-expression of class I HLA antigens and beta-2-m was therefore studied. All three classes (alpha, beta, and gamma) of IFN were capable of re-inducing HLA and beta-2-m, although gamma-IFN was more potent. Of a variety of chemical differentiating agents, only 1,25-dihydroxyvitamin D3 (calcitriol) was found to induce HLA class I antigens. These results suggest that HLA and beta-2-m are not necessarily constitutive cell surface proteins, but instead that their expression is highly inducible.

Antibodies, Monoclonal↗
PubMed1984

Differentiation of a human monocytic cell line by 1,25-dihydroxyvitamin D3 (calcitriol): a morphologic, phenotypic, and functional analysis.

Several recent studies have suggested a role for 1,25-dihydroxyvitamin D3 (calcitriol) in myeloid differentiation. We have examined the effects of calcitriol on the U937 monoblast cell line and found that calcitriol, at near physiologic concentrations, is a potent inhibitor of U937 growth. Moreover, calcitriol induces differentiation to a monocyte-macrophage phenotype marked by enhanced alpha-naphthyl esterase staining. Morphologic changes were attended by de novo induction of the myeloid specific antigen detected by the monoclonal antibody AML-2-23, as well as dramatic increases in Fc receptors for IgG. In addition, calcitriol induced U937 cells to perform phagocytosis and antibody-dependent cellular cytotoxicity. These results indicate the potential activity of calcitriol in myeloid differentiation and additionally suggest a role for calcitriol in monocyte-macrophage activation.

Antibodies, Monoclonal↗
PubMed1983

Cytofluorographic analysis of receptors for IgA on human polymorphonuclear cells and monocytes and the correlation of receptor expression with phagocytosis.

Receptors for the Fc portion of IgA (RFc alpha) were identified on human monocytes and polymorphonuclear cells (PMNs) by cytofluorograph analysis using FITC-labeled IgA and by rosette formation with IgA-sensitized indicator cells. Larger percentages of PMNs obtained from the oral cavity expressed RFc alpha than did blood PMNs. These oral PMNs also expressed more RFc alpha per cell than blood PMNs. Moreover, the expression of these receptors on blood PMNs was enhanced by overnight incubation with IgA. In functional studies, RFc alpha appeared to cooperate with receptors for IgG in enhancing blood and oral PMN mediated phagocytosis of target cells coated with IgG and IgA. Furthermore, and in contrast with blood PMNs, oral PMNs were capable of phagocytosing target cells coated with IgA alone. Thus, receptors for IgA may be important to the ability of RFc alpha bearing effector cell populations to mediate protection at mucosal surfaces.

Cells, Cultured↗
PubMed1983

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.

Antibodies, Monoclonal↗
PubMed1983

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.

Animals↗
PubMed1983

The use of myeloid-specific monoclonal antibodies in the diagnosis of leukemia.

Leukemia cells express a variety of cell surface antigens that have been useful in the delineation of subclasses of both lymphocytic and myeloid leukemia. In this report we describe the phenotype of cells from 50 patients with leukemia based on the results of testing for binding to four monoclonal antibodies reactive with myeloid cell-associated antigens and, in some cases, three monoclonal antibodies reactive with lymphocyte-associated antigens. We related the patterns of reactivity of these leukemia cells with the diagnosis based on morphology and histochemistry. Three of the myeloid cell-associated antigens (PMN 6, PMN 29, and AML-2-23) were expressed only on cells from patients with AML. The fourth, PM-81, was expressed on cells from 29/31 patients with AML and on 3/5 with ALL. The reactivity of the ALL cells with the J5 and B1 antibodies confirmed the diagnosis of ALL. A proposed schema for the use of these monoclonal antibodies in both the diagnosis and subclassification of leukemia is described. Finally, the significance of the heterogeneity in antigen density on leukemia cells is considered.

Animals↗
PubMed1983

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody.

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.

Animals↗
PubMed1983

Monoclonal antibodies to myeloid differentiation antigens: in vivo studies of three patients with acute myelogenous leukemia.

Three patients with acute myelogenous leukemia (AML) in relapse were treated with intravenous infusions of one or more purified murine monoclonal antibodies (MoAbs) specific for differentiation antigens on normal and malignant myeloid cells. Three of the MoAbs used were IgM immunoglobulins that react with glycolipids, while the fourth, an IgG2b, reacts with a protein antigen. Peripheral blood leukemia cell counts decreased significantly, but transiently, during treatment. Evidence of in vivo binding of each MoAb to leukemia cells was obtained, although two of the four MoAbs could not be detected in the plasma following infusion, perhaps due to circulating blocking factors. Antigenic modulation was not encountered in these studies. However, the induction of human antibody to murine MoAb was observed in one patient who was treated over a 70-day period. Toxicities encountered were minimal and included fever (3 patients), back pain (1 patient), and arthralgias and myalgias (1 patient). This is the first reported clinical trial of (1) IgM MoAbs, (2) MoAb therapy in patients with AML, (3) combinations of MoAbs directed toward different myeloid differentiation antigens, and (4) MoAbs directed to glycolipids. The relative lack of toxicity and the positive effects of MoAb treatment in the reduction of leukemia cell counts permit the continued study of more innovative approaches to the treatment of AML with MoAbs.

Adult↗
PubMed1982

Monoclonal antibodies to novel myeloid antigens reveal human neutrophil heterogeneity.

Three cytotoxic murine monoclonal antibodies that recognize myeloid-specific antigens have been produced by immunization with normal human neutrophils or myeloblasts from a patient with acute myelomonocytic leukemia. Two of these, PMN 6 and PMN 29, are specific for neutrophils; the third monoclonal antibody, AML-2-23, is reactive with the majority of normal monocytes as well as a subpopulation of mature neutrophils. Although neutrophils from all individuals tested expressed these antigens, cytofluorographic analysis revealed that the percentage of cells bearing the PMN 6 and AML-2-23 antigens varied among individuals. Significant additional heterogeneity in the density of each antigen among antigen-bearing cells was also observed. All three antibodies efficiently mediated complement-dependent cytotoxicity of acute myelocytic leukemia cells yet were unreactive with lymphocytic leukemia cells. Neutrophil cytotoxicity was mediated by PMN 6 and PMN 29 but not by AML-2-23. On the other hand, AML-2-23, but not PMN 6 or PMN 29, was cytotoxic for normal monocytes and macrophages. These monoclonal antibodies may be of value in the study of normal neutrophil function and differentiation and may have clinical utility in diagnosis and therapy of myeloid leukemia.

Animals↗
PubMed1982

Studies on the ability of monoclonal antibodies to selectively mediate complement-dependent cytotoxicity of human myelogenous leukemia blast cells.

A panel of monoclonal antibodies that bind to leukemic blast cells from patients with acute myelocytic leukemia and chronic myelocytic leukemia in blast crisis was studied for their ability to mediate complement-dependent lysis of a variety of cell populations from patients with leukemia, normal blood cells, and human leukemia cell lines. Several of these monoclonal antibodies were selectively cytotoxic to myeloid leukemia cells (AML-1-99, AML-1-211, AML-2-30, CML-75, CML-115, and CML-150). Although they were all capable of binding to normal cell populations, none of these hybridomas were cytotoxic to normal cells. Three of these antibodies (AML-1-211. CML-75, and CML-150) were cytotoxic to some leukemia cell samples only after dilution of the hybridoma supernatant, i.e., they showed a prozone. Binding of these three antibodies, as well as another, AML-1-201, as determined in a radioimmunoassay, also showed a prozone. Other monoclonal antibodies are described (AML-2-23 and AML-2-9) that mediate complement-dependent cytotoxicity to myeloid leukemia cells as well as selected normal cell types (monocytes and lymphocytes, respectively). The potential clinical utility of these monoclonal antibodies is considered in the context of recently encountered problems in the use of monoclonal antibodies to mediate leukemia cell lysis in vivo.

Animals↗