PubMed Health⌕ Search

Biomedical subjects

M W Fanger

Publications and source records attributed to M W Fanger.

At least 73 records · Page 4Linked to original sources
PubMed1989

Cytotoxicity mediated by human Fc receptors for IgG.

The Fc receptors for IgG(Fc gamma R) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of Fc gamma R-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these Fc gamma R in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monoclonal antibodies specific for Fc gamma R on human leukocytes has established the existence of three distinct Fc gamma R and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of Fc gamma R-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different Fc gamma R is discussed. The results of these studies suggest that the ability of a given Fc gamma R to trigger killing is sometimes dependent on the type of Fc gamma R, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.

Animals↗
PubMed1987

Fc gamma RI and Fc gamma RII on monocytes and granulocytes are cytotoxic trigger molecules for tumor cells.

As part of an effort to define the cytotoxic trigger molecules on human myeloid cells, the ability of the different Fc receptors for IgG (Fc gamma R) to mediate killing of tumor cell lines by monocytes and granulocytes was examined. This was accomplished by studying cytolysis of hybridoma cell (HC) targets bearing surface antibody directed toward the different Fc gamma R. The HC line, HC IV.3A, which bears Ig directed to the low affinity Fc gamma R (Fc gamma RII) on monocytes and neutrophils was lysed by human monocytes. The extent of lysis of HC IV.3A was approximately equal to that of anti-Fc gamma RI (the high affinity Fc gamma R on human monocytes) bearing HC lines (HC 32.2A and HC 62A) and was not augmented by treatment of the monocytes with interferon-gamma (IFN-gamma). In contrast, neutrophils lysed HC IV.3A and HC 32.2A only after activation with IFN-gamma. Since Fc gamma RI is not detectable on untreated neutrophils and is induced by IFN-gamma on these cells, lysis of HC 32.2A by IFN-gamma-activated neutrophils correlated with receptor induction. On the other hand, Fc gamma RII was present at equal levels on untreated and IFN-gamma-treated neutrophils, but only IFN-gamma-treated neutrophils mediated cytotoxicity via Fc gamma RII. In this case, enhanced killing appeared to be due to events other than an increase in Fc gamma RII number. Neither untreated nor IFN-gamma-treated neutrophils mediated the lysis of the anti-Fc gamma RIII bearing HC 3G8A. Thus, binding to the tumor target via this Fc receptor does not lead to lysis and may initiate signals distinct from those triggered through Fc gamma RI or Fc gamma RII. Surprisingly, HC bearing high amounts of mouse IgG1 antibody of irrelevant specificity were also lysed by monocytes. This lysis was blocked by soluble IV.3 antibody and thus appeared to be due to binding of the Fc portion of the surface Ig to Fc gamma RII on monocytes. Furthermore, monocytes from donors with a form of Fc gamma RII incapable of binding aggregated mouse IgG1 did not lyse these HC, but displayed normal lysis of HC IV.3, demonstrating that this structurally different Fc gamma RII remained a functional trigger molecule. Overall, these studies have demonstrated the specificity of Fc receptors in triggering monocyte- and granulocyte-mediated antibody-dependent tumor cell killing and have begun to dissect functional similarities and differences among the three defined Fc gamma R on human myeloid cells.

Animals↗
PubMed1987

Polymorphonuclear leukocyte function triggered through the high affinity Fc receptor for monomeric IgG.

While it is extensively documented that gamma-interferon (IFN-gamma) is a potent stimulator of cells of the monocyte lineage, relatively little is known about its effects on granulocytes. We and others have found that immunoglobulin G (IgG) antibody-dependent cell cytotoxicity (ADCC) by polymorphonuclear cells (PMN) is significantly enhanced in a dose-dependent fashion by 16 hours incubation with recombinant IFN-gamma, resulting in 2- to 16-fold increases in ADCC. Incubation of PMN with lipopolysaccharide for 16 hours did not augment ADCC. Since IFN-gamma enhancement of ADCC is accompanied by increased expression of Fc receptors, we used monoclonal antibodies to compare control and IFN-gamma treated PMN for expression of the high affinity Fc receptor for monomeric IgG1 (FcgRI) and the PMN receptor for polymeric IgG (FcgR1o). Freshly isolated PMN or PMN cultured without IFN-gamma expressed FcgR1o but not detectable quantities of FcgRI. However, while FcgR1o were not increased on IFN-gamma-treated PMN, these cells expressed moderate amounts of FcgRI. To determine whether FcgRI contributed to PMN function, heteroantibodies consisting of Fab 3G8 or Fab 32 linked to Fab anti-target antibody were produced. ADCC of untreated PMN was promoted only by Fab 3G8 heteroantibody, whereas IFN-gamma-treated PMN killed through both FAB 3G8 and Fab 32 heteroantibodies. Thus, FcgRI can be induced on PMN by IFN-gamma, can mediate cytotoxicity by these cells, and probably accounts for the IFN-gamma stimulation of ADCC.

Antibodies, Monoclonal↗
PubMed1987

Human monocyte-mediated cytotoxicity: the use of Ig-bearing hybridomas as target cells to detect trigger molecules on the monocyte cell surface.

We recently reported the preparation and characterization of a monoclonal antibody, 32.2, specific for the high-affinity Fc receptor (FcR) for IgG on human monocytes. We have utilized the hybridoma cell line producing this antibody as a target for monocyte-mediated cytotoxicity. The hybridoma was selected for stable sublines that expressed high quantities of surface 32.2 immunoglobulin (Ig) through flow cytometry. Monocyte-mediated cytotoxicity, with these sublines used as targets, was evaluated with the use of a 51Cr-release assay. It was found that monocytes could efficiently lyse the hybridoma cells (HC 32.2) bearing surface Ig directed to the high-affinity FcR. Consistent with the specificity of the 32.2 antibody for an epitope on the high-affinity receptor outside of the ligand binding site, human IgG did not block monocyte killing of HC 32.2. In contrast, monocytes could not mediate lysis of hybridoma cells bearing high levels of antibody directed to other monocyte cell surface molecules, in particular, class I MHC molecules, the C3bi receptor, and the My 23 antigen. The effect of IFN-gamma on the ability of monocytes to mediate lysis of the 32.2 Ig-bearing hybridomas was also assessed. Monocytes cultured in the absence of IFN-gamma could lyse the hybridoma line expressing high levels of 32.2 Ig as efficiently as monocytes cultured in the presence of IFN-gamma. However, untreated monocytes were less able than IFN-gamma-treated monocytes to kill HC 32.2 expressing lower levels of Ig. Thus, IFN-gamma may enhance the efficiency of monocyte-mediated antibody-dependent killing under conditions where limited antibody is available on the target. These studies demonstrate that the high-affinity FcR on monocytes can act as a cytotoxic trigger molecule for killing of tumor cell targets and that this trigger does not require specific binding to the Fc binding epitope. These results further encourage possible clinical application of the 32.2 monoclonal antibody in tumor therapy.

Animals↗
PubMed1987

Comparison of the effects of 1,25-dihydroxyvitamin D3 on T lymphocyte subpopulations.

Previous studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol) is a potent inhibitor of human T lymphocyte proliferation. It has been reported that only CD4+ cells are sensitive to the anti-proliferative action of calcitriol. To further evaluate this observation, we first performed cell cycle analysis of unfractionated phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) in the absence or presence of calcitriol. The CD4/CD8 ratio was similar between control and treated cells for each phase of cycle (G0----G1, S, G2 + M), suggesting that calcitriol did not selectively block proliferation of either T cell subpopulation. Secondly, the growth-inhibitory activity of calcitriol on PBMC selectively depleted of either CD4+ or CD8+ cells was comparable to that observed with unfractionated PBMC. Furthermore, the induction of transferrin receptors was inhibited by calcitriol to a comparable degree in each T cell subset, suggesting that equivalent inhibition of transition into late G1 was observed. Finally, calcitriol inhibited the proliferation of highly purified T cell subsets (greater than 99% pure) to equivalent degrees. These data suggest that T cell subsets defined by either the CD4 or by the CD8 antigen are both sensitive to the growth inhibitory effects of calcitriol.

Antibodies, Monoclonal↗
PubMed1987

Monoclonal antibodies that bind to the My23 human myeloid cell surface molecule: epitope analysis and antigen modulation studies.

It has previously been shown that the AML-2-23 monoclonal antibody (MoAb) reacts with a glycoprotein on differentiated myeloid cells. The antigen, My23, is released from these cells in culture and is also detectable in normal human plasma. We have now raised a panel of MoAbs against the soluble form of the antigen which reacts with monocytes and calcitriol-treated U937 and HL-60 myeloid cell lines, but not with lymphocytes or undifferentiated U937 and HL-60 cells. As with the AML-2-23 MoAb, the anti-My23 MoAbs immunoprecipitated a 50,000-55,000 protein from calcitriol treated HL-60 cells. Besides binding to cell surface My23, all eight MoAbs as well as the 63D3 MoAb reacted with crude and purified forms of soluble My23. A novel ELISA epitope analysis assay was developed to identify four distinct antigenic determinants on My23. Thus, the soluble and cell surface forms of My23 share several antigenic determinants and are biochemically very similar. Pooled anti-My23 caused selective patching, capping and clearing of My23 from the cell surface. My23 was not associated with cell surface, HLA-DR, HLA-A,B,C, beta 2-microglobulin or the Fc receptors for IgG or IgA. These anti-My23 MoAbs should be of importance in antigen functional studies and in clinical treatment protocols for patients with acute myelogenous leukemia. Furthermore, we have shown that a soluble myeloid differentiation antigen can serve as an effective immunogen for the preparation of MoAbs against important cell surface molecules thus obviating many problems inherent in the use of whole cells or detergent lysate-derived immunoprecipitates as immunogens.

Animals↗
PubMed1987

Regulation of lymphokine production and human T lymphocyte activation by 1,25-dihydroxyvitamin D3. Specific inhibition at the level of messenger RNA.

The steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (calcitriol), has been shown to inhibit T cell proliferation, primarily through inhibition of interleukin 2 (IL-2) production. In these experiments, we show that calcitriol also markedly inhibited production of the lymphokine, gamma interferon (IFN-gamma), by activated human T lymphocytes. Regulation of both IL-2 and IFN-gamma production as well as transferrin receptor (TfR) expression by calcitriol was apparent at the messenger RNA (mRNA) level as determined by Northern blotting. The decrease in IL-2 and IFN-gamma mRNA that occurred with calcitriol treatment was coordinate and not apparent up to 12 h after phytohemagglutinin stimulation, whereas decreased accumulation of TfR mRNA was not present before 24-36 h. Furthermore, the effects of calcitriol on IL-2, IFN-gamma, and TfR mRNA accumulation were specific; actin mRNA accumulation was comparable between control and treated cells. These data indicate that calcitriol regulated proteins associated with T cell activation at the transcriptional level and that these effects were mediated in a specific, coordinate fashion.

Actins↗
PubMed1986

Heteroantibody-mediated cytotoxicity: antibody to the high affinity Fc receptor for IgG mediates cytotoxicity by human monocytes that is enhanced by interferon-gamma and is not blocked by human IgG.

An IgG1 monoclonal antibody, 32.2, raised against the 72,000 dalton monocyte high affinity Fc receptor, was used to examine the role of this receptor in ADCC. This antibody did not inhibit the binding of human IgG1 to monocytes or to the U937 cell line, nor did it block or stimulate their killing of IgG-coated chicken erythrocytes (CE). Whole 32.2 or its Fab fragments were cross-linked to Fab fragments of rabbit anti-CE by using the agent SPDP. The resulting heteroantibodies (32.2 X Fab anti-CE) mediated monocyte and U937 cytotoxicity against CE, whereas an anti-HLA X anti-CE reagent did not. Both FcR expression and heteroantibody-mediated cytotoxicity were increased by culturing monocytes or U937 with IFN-gamma. Although IgG-mediated ADCC was significantly inhibited by 40 micrograms/ml human IgG1, cytotoxicity mediated by 32.2 X Fab anti-CE was not blocked by 2 mg/ml human IgG1, suggesting that such cytotoxicity might not be blocked by IgG in vivo. These data indicate the potential of 32.2 heteroantibodies in analysis of FcR function and in therapy.

Antibodies, Monoclonal↗
PubMed1986

Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line.

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.

Antibodies, Monoclonal↗
PubMed1986

Lysis of small cell carcinoma of the lung tumor cell lines by gamma interferon-activated allogeneic peripheral blood mononuclear cells: abrogation of killing by pretreatment of tumor cells with gamma interferon.

Interferon has been shown to enhance the ability of nonspecific cytotoxic mononuclear cells to lyse some, but not all, tumor cells. We have examined the effect of recombinant human gamma interferon (rIFN gamma) on the cell-mediated cytolysis of tumor target cells derived from continuously cultured lines of small cell carcinoma of the lung (SCCL). Cells from the SCCL lines DMS 44, 53, 79, 92, and 406 were labeled with 51Cr and incubated with normal and rIFN gamma-stimulated peripheral blood mononuclear cells for 18 h at 37 degrees C and tumor cell lysis estimated by measuring 51Cr release. Although cells from certain SCCL lines were good targets for cell mediated cytotoxicity, susceptibility to lysis was heterogeneous among the different SCCL lines. DMS 406 and 79 were, on average, maximally lysed, while DMS 44, 53, and 92 showed less susceptibility to lysis by either control or rIFN gamma-stimulated effector cells. In addition, although pretreatment with rIFN gamma increased the cytolytic capacity of normal peripheral blood mononuclear cells from several different donors, preincubation of the tumor cell lines with rIFN gamma resulted in inhibition of cytolysis mediated by both control and IFN-activated effector cells. These findings suggest that although rIFN gamma may enhance cell-mediated lysis of SCCL tumor cells, it may also decrease susceptibility to lysis.

Antigens, Surface↗
PubMed1986

IgA-mediated effector function of HL-60 cells following treatment with calcitriol.

When cells of the HL-60 promyelocytic leukemia line are cultured with 1,25-dihydroxyvitamin D3 (calcitriol) they acquire a more highly differentiated, myelomonocytic phenotype. It was observed that the ability to ingest IgA-coated erythrocytes and to bind soluble dimeric IgA accompanied this maturation. Phagocytosis of IgA-coated erythrocytes was greater than 50% inhibited by 0.8 mg/ml free IgA, and not by IgG or IgM. Similarly, binding of dimeric IgA was not blocked by a 100-fold excess of IgG, IgM or IgE. Both IgA-mediated phagocytosis and IgA binding became apparent after two days of culture with calcitriol and increased with time in culture. The induction of functional IgA receptors was evident with 10(-11) M calcitriol and maximal levels of IgA binding and of numbers of cells capable of IgA mediated phagocytosis were induced by 10(-8)-10(-9) M calcitriol. 25-Hydroxyvitamin D3, which binds 100-1000-fold less avidly to the cytoplasmic D3 receptor than calcitriol, did not induce functional IgA receptors unless concns of 10(-7) M were used. Other compounds which induce differentiation of HL-60 cells, including retinoic acid and DMSO, produced similar results to calcitriol, whereas cells treated with gamma interferon expressed lower levels of IgA binding and did not ingest IgA-coated targets, suggesting that a critical density of IgA receptors must be reached to enable phagocytosis and/or that other cell activational events are required for IgA receptors to mediate killing. This model may provide useful insight into the function and regulation of IgA receptors on cells of the myeloid series.

Antigens, CD↗
PubMed1986

Glucocorticoid enhances gamma interferon effects on human monocyte antigen expression and ADCC.

The expression of HLA-DR antigen by highly enriched human monocytes cultured in serum free medium was found to be markedly elevated by human recombinant gamma interferon (IFN-gamma). This effect was maximal after 48 h in culture with 300 mu/ml IFN-gamma. Class I MHC antigen also increased with IFN-gamma treatment. By contrast, binding of a myeloid-specific monoclonal antibody, AML-2-23, was dramatically decreased by IFN-gamma. The augmentation of MHC antigens was not ablated by an immunosuppressive concentration (2 X 10(-7) M) of the glucocorticoid dexamethasone (DEX). In fact, both the enhancement of Class I and Class II MHC antigen expression and the suppression of AML-2-23 antigen by IFN-gamma were often more profound in the presence of DEX. IFN-gamma treatment also resulted in elevated monocyte effector function, as measured by antibody dependent cellular cytotoxicity (ADCC). This functional activation was not inhibited by DEX. On the contrary, DEX slightly augmented IFN-gamma effects on ADCC. This contrasts with other reports that glucocorticoids inhibit monocyte responsiveness to lymphokines, and suggests that the interplay between lymphokines and the glucocorticoid hormones may be more complex than previously thought.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1985

1,25-Dihydroxyvitamin D3 induces a myelomonocytic phenotype with enhanced effector cell function in the HL-60 promyelocytic leukemia cell line.

Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.

Antibodies, Monoclonal↗
PubMed1985

The effects of recombinant-DNA-derived interferons on the growth of myeloid progenitor cells.

Interferons (IFNs) have been shown to have significant effects on hematopoietic cell growth. Previous studies defining these effects have utilized mouse and human alpha-, beta-, and gamma-IFN isolated from supernatants of stimulated cells. Despite purification, the possible presence of other lymphokines and soluble factors remains a concern. In this study, the effects of gene-cloned alpha- and gamma-IFN on colony-forming units of granulocyte/macrophage (CFU-GM) progenitors cultured from the peripheral blood of normal volunteers were examined. In addition, blast cell colonies from one patient with acute myelogenous leukemia (AML) were studied. The growth of normal CFU-GM and AML blast cell colonies was inhibited in a dose-dependent manner by gamma- and alpha-IFN. gamma-IFN was ten to 100 times more potent than alpha-IFN in that this species of IFN reduced colony formation by greater than 50% at concentrations of less than 15 antiviral U/mL. The effects of gamma-IFN were neutralized by a monoclonal antibody specific for gamma-IFN. These in vitro studies indicate that human gamma-IFN may be an important modulator of myelopoiesis. Although these data indicate a possible efficacy of gamma-IFN in the treatment of AML, the in vitro results should be considered for their in vivo significance.

Clone Cells↗
PubMed1985

The effects of 1,25-dihydroxyvitamin D3 on human T lymphocyte activation and proliferation: a cell cycle analysis.

Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, is a potent inhibitor of both lectin- and antigen-driven human T lymphocyte proliferation. To better characterize this effect, we performed cell cycle analysis of both untreated and calcitriol-treated peripheral blood mononuclear cells after PHA stimulation. By using the metachromatic dye acridine orange and flow cytometry, we found that calcitriol blocks the transition from the early, low RNA compartment of G1 (G1A) to the late, higher RNA compartment of G1 (G1B). Consistent with this observation was the inability of exogenous IL 1 or phorbol myristic acetate to overcome calcitriol's suppression of DNA synthesis. Indomethacin slightly reversed calcitriol's inhibition of transition from early to late G1, suggesting a minor, prostaglandin-dependent component to calcitriol's antiproliferative activity. Finally, by using the monoclonal antibodies anti-Tac and OKT9, we found that calcitriol had no effect on IL 2 receptor expression, an early G1 event, but markedly inhibited transferrin receptor expression, an IL 2-dependent, late G1 event. Thus, analysis of calcitriol's effects on the expression of these T cell activation antigens provides further evidence of the cell cycle specificity of calcitriol's action in regulating human T lymphocyte proliferation.

Calcitriol↗
PubMed1985

The expression of receptors for IgA on human monocytes and calcitriol-treated HL-60 cells.

In this study, we investigated the expression of IgA Fc receptors (FcR alpha) by human myeloid cells. By using a sensitive cytofluorometric IgA binding assay, we found that approximately 85% of peripheral blood monocytes bound IgA in an isotype-specific fashion. The HL-60 human promyelocytic leukemia cell line failed to express FcR alpha; however, treatment of HL-60 cells with the differentiating agent calcitriol induced the expression of FcR alpha on greater than 90% of these cells. Monocytes and calcitriol-treated HL-60 cells were capable of ingesting IgA-coated erythrocyte targets, suggesting that phagocytosis could be mediated through FcR alpha. The induced HL-60 cell system represents a useful model for further studies on FcR alpha expression and function.

Antigens, CD↗