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.

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.

Monocytes and polymorphonuclear neutrophils of patients with streptococcal pharyngitis express increased numbers of type I IgG Fc receptors.

Studies using cultured cells have shown that gamma interferon (IFN-gamma) induces the expression of Fc gamma RI (the type I Fc receptor for IgG) on human polymorphonuclear neutrophils (PMN) and greatly increases the number of these receptors on human monocytes. Administration of rIFN-gamma in vivo also causes enhanced Fc gamma RI expression on these cell populations. Because streptococcal antigens are potent inducers of IFN-gamma in vitro, we postulated that IFN-gamma would be produced endogenously in vivo in patients with streptococcal infections. Such production of IFN-gamma in vivo, even at low levels, might be expected to induce the expression of Fc gamma RI on monocytes and neutrophils. To evaluate this possibility, we used monoclonal antibody 32 (mAb 32), which is specific for Fc gamma RI, to quantitate the expression of this receptor on human peripheral blood cells. We measured the binding of mAb 32 to monocytes and PMNs isolated from healthy donors and from patients with group A beta-hemolytic streptococcal (GABHS) pharyngitis. PMNs from healthy donors (n = 12) had 700 +/- 600 (mean +/- SD) mAb 32 binding sites. Patients with pharyngitis and negative throat culture for GABHS (n = 11) had 2,100 +/- 1,600 sites on their PMNs. In contrast, the PMNs from patients with documented GABHS pharyngitis (n = 12) had 11,600 +/- 7,500 mAb 32 binding sites on their surface. There was a similar change in the expression of Fc gamma RI on monocytes, with control monocytes having a mean of 19,900 +/- 3,200 mAb 32 binding sites per cell and the GABHS-positive monocytes having 47,500 +/- 21,400 sites. The GABHS-negative throat culture group had a slightly elevated number of Fc gamma RI with a mean of 28,200 +/- 8,400 sites. 10 patients with documented urinary tract infections and three patients with uncomplicated pyelonephritis had no elevation in Fc gamma RI expression. These studies demonstrate that a localized group A streptococcal infection can cause systemic activation of the entire circulating pool of phagocytes, and suggest that a similar level of activation is uncommon in localized gram-negative infections of the urinary tract.

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.

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.

Bispecific antibody-dependent cellular cytotoxicity of HER2/neu-overexpressing tumor cells by Fc gamma receptor type I-expressing effector cells.

A bispecific antibody, MDX-H210, was developed to target cytotoxic effector cells expressing Fc gamma receptor type I (Fc gammaRI, CD64) to HER2/neu-overexpressing tumor cells. HER2/neu is an appropriate target for immunotherapy due to the high level of expression of this proto-oncogene in a variety of malignancies. The expression of Fc gammaRI is limited primarily to cytotoxic immune cells, including monocytes, macrophages, and cytokine-activated polymorphonuclear (PMN) cells. Therefore, tumor cells bound with MDX-H210 can be selectively recognized by effector cells with cytotoxic potential. MDX-H210 was prepared by chemical conjugation of Fab' fragments derived from the HER2/neu-specific monoclonal antibody, 520C9, and the Fc gammaRI-specific monoclonal antibody, H22. This bispecific molecule demonstrated specific, dose-dependent, and saturable binding to both HER2/neu- and Fc gammaRI-expressing cells. A solid-phase immunoassay that demonstrated simultaneous and specific binding to both antigens was used to confirm the bispecific nature of MDX-H210. Monocytes and PMN cells mediated MDX-H210-dependent lysis of HER2/neu-overexpressing cell lines derived from breast, ovarian, and lung carcinomas. IFN-gamma treatment of monocytes enhanced antibody-dependent cellular cytotoxicity, whereas IFN-gamma and granulocyte colony-stimulating factor were required for PMN cell-mediated tumor cell lysis. In addition, MDX-H210 elicited tumor necrosis factor-alpha secretion from monocytes when cultured in the presence of HER2/neu-positive target cells. These in vitro data suggest that targeting tumor cells to Fc gammaRI with MDX-H210 may be an effective treatment for malignancies that overexpress HER2/neu. The in vivo cytotoxic potential of MDX-H210 may be enhanced by combination therapy with the cytokines granulocyte colony-stimulating factor and IFN-gamma, which up-regulate Fc gammaRI expression on cytotoxic effector cells.

Bispecific antibodies.

Bispecific antibodies--molecules combining two different antigenic specificities--are currently being developed as new agents for immunotherapy and for basic studies in cell biology. Bispecific antibodies (BsAb) are prepared by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid-hybridoma. Both of these approaches present challenges with respect to yield and purity that should eventually be solved through newer molecular genetic approaches. BsAb have been used to demonstrate that specific surface molecules can trigger leukocytes to either phagocytose or kill tumor cells, viruses, parasites, and infected cells. Such trigger molecules include CD3 on T lymphocytes and Fc receptors for IgG on monocytes, macrophages, and natural killer cells. BsAb have also been used experimentally to localize toxins to tumor sites and fibrinolytic agents to areas of thrombosis, to study the molecular specificity of particular receptors, and as adjuvants in in vitro models of vaccines for infectious disease. The limited clinical trials that have occurred to date, primarily for therapy of tumors, suggest that BsAb may offer considerable promise for therapeutic applications, including cancer, heart disease, infectious disease, allergy, and autoimmunity.

Targeting HIV-1 to Fc gamma R on human phagocytes via bispecific antibodies reduces infectivity of HIV-1 to T cells.

In addition to CD4, the primary receptor to which the human immunodeficiency virus type 1 (HIV-1) binds, mononuclear phagocytes (monocytes) express three classes of Fc receptors for immunoglobulin G (Fc gamma R). We have previously shown that infection of monocytes by HIV-1 is inhibited when bispecific antibodies (BsAbs) are used to target the virus to either the type I, type II, or type III Fc gamma R on these cells. Infection of monocytes was not inhibited when HIV-1 was targeted to either human leukocyte antigen class I or CD33. We have extended these studies to examine the ability of BsAbs plus polymorphonuclear leukocytes (neutrophils, PMNs) and monocytes to reduce infectivity of HIV-1 to cells from the human T cell lymphoma line, H9. The production of HIV-1 following interaction of virus with BsAb and phagocytes was determined in an indicator cell assay by mixing BsAb, HIV-1, and phagocytes with uninfected H9 cells. Productive infection of H9 cells was quantitated on subsequent days by measuring p24 gag antigen levels in supernatants by enzyme-linked immunosorbent assay. Our findings show that the addition of interferon-gamma-activated PMNs or monocytes to cultures of HIV-1 plus H9 cells in the absence of BsAb results in a marked reduction in p24 levels equivalent to 85 to 90% of control levels. With the combination of BsAb (anti-Fc gamma RI x anti-gp120) plus IFN-gamma-activated phagocytes, levels of p24 in H9 cultures were below those at culture initiation. These findings demonstrate that IFN-gamma-activated phagocytes can affect the natural course of HIV-1 infection of T cells, a finding of potential clinical importance.

Role of Fc gamma receptors in cancer and infectious disease.

Receptors for the Fc domain of immunoglobulin G (Fc gamma R) provide an interface between specific humoral immunity and the cellular branch of the immune system through their interaction with antibody. Cross-linking Fc gamma R on myeloid cells triggers such diverse functions as clearance of immune complexes, phagocytosis of opsonized pathogens, secretion of reactive oxygen intermediates, and antibody-dependent cellular cytotoxicity. The Fc gamma R play a major role in the removal of antibody-coated infectious agents and are the exclusive trigger molecules for tumor cell killing by human myeloid cells. Studies of Fc gamma R function have been aided by the use of Fc gamma R specific monoclonal antibodies, self-directed target cells, and bispecific antibodies that link target cells or pathogens to specific host cell molecules, including Fc gamma R. These reagents have contributed significantly to our understanding of the role of the different classes of Fc gamma R in mediating protection from various infectious agents and in mediating tumor cell killing. Taken together, these approaches have provided insight into the utility of manipulating Fc gamma R function in the therapy of cancer and infectious disease.

Humanized mAb H22 binds the human high affinity Fc receptor for IgG (FcgammaRI), blocks phagocytosis, and modulates receptor expression.

About 10-15% of patients with immune thrombocytopenic purpura (ITP) cannot be controlled by corticosteroid therapy and splenectomy. For these patients treatment with high-dose IVIgG induces partial or complete responses. The clinical benefits of IVIgG could be due to blockade of Fc receptors for IgG (FcgammaR), because several model systems clearly show that functional FcgammaR are essential for establishment of ITP and related diseases. However, the specific contributions of the three individual classes of FcgammaR remain to be more completely defined. Recently monoclonal antibody (mAb) H22, which recognizes an epitope on FcgammaRI (CD64) outside the ligand binding domain, was humanized by grafting its complementarity determining regions onto human IgG1 constant domains. Because FcgammaRI has a high affinity for human IgG1 antibodies, we predicted mAb H22 would also bind to FcgammaRI through its Fc domain and block FcgammaRI-mediated phagocytosis. These studies demonstrate that mAb H22 blocked phagocytosis of opsonized red blood cells 1000 times more effectively than an irrelevant IgG. Moreover, cross-linking FcgammaRI with mAb H22 rapidly down-modulated FcgammaRI expression on monocytes without affecting other surface antigens. We conclude that because mAb H22 is a humanized mAb that blocks the FcgammaRI ligand binding domain and down-modulates FcgammaRI expression, it is a particularly good candidate for evaluating the role of FcgammaRI in patients with ITP.

Unique CD8+ T cell-rich lymphoid aggregates in human uterine endometrium.

Using confocal scanning laser microscopy of viable tissue sections, we have demonstrated organized lymphoid aggregates (LA), that have a unique structure, in the stratum basalis of uterine endometrium. These LA consist of a core of B cells surrounded by more numerous T cells and an outer halo of monocytes/ macrophages. The T cells in the LA were almost exclusively CD8+CD4-. These CD8+ LA, in terms of both their T cell and B cell components, were either small or absent during the early proliferative stage of the menstrual cycle, significantly larger in size at mid-cycle and during the secretory phase, and absent in post-menopausal women, suggesting that their development is hormonally influenced. This new finding of a menstrual cycle-dependent, phenotypically unique, organized immune cell structure may lead to new insights into the mechanisms by which the endometrium accepts a semiallogeneic graft while providing resistance to infectious organisms.

The functional properties of Fc gamma RI, II and III on myeloid cells: a comparative study of killing of erythrocytes and tumor cells mediated through the different Fc receptors.

Three distinct Fc receptors for IgG, Fc gamma RI, Fc gamma RII and Fc gamma RIII are known to be associated with human myeloid cells. Using mAb specific for these receptors, and the hydridoma cells lines that produce these mAb, we have examined the ability of each of these receptors on different myeloid cells and cell lines to mediate killing of tumor and red cell targets. Hybridoma cells (HC) expressing anti-Fc gamma RI, Fc gamma RII or Fc gamma RIII upon their surface were used as model self-directed tumor targets. Chicken erythrocytes (CE) were used as another type of target cell and in this case effector cell cytotoxicity was mediated by heteroantibodies (HA) composed of Fab fragments of anti-Fc gamma R mAb covalently linked to Fab fragments of rabbit anti-CE antibodies. Monocytes, lymphocytes, polymorphonuclear cells (PMNs) and the myeloid cell lines U937, HL-60 and THP-1 were used as effector cells either in their native state or after activation with rIFN-gamma. Direct comparison of cytotoxicity by the same effector cell population against both tumor and erythroid targets has permitted definitive evaluation of the ability of the different Fc gamma R to promote cytolysis under two different conditions. Monocytes were able to utilize Fc gamma RI, Fc gamma RII and Fc gamma RIII in killing both CE and HC targets, and incubation with rIFN-gamma augmented their ability to kill CE, particularly through Fc gamma RI. Fc gamma RII and Fc gamma RIII mediated killing of CE by untreated neutrophils. rIFN-gamma induced PMNs to express Fc gamma RI and to mediate killing of CE through this receptor. Moreover, HC targets were not lyzed by untreated neutrophils, but rIFN-gamma activated neutrophils killed HC bearing surface anti-Fc gamma RI and anti-Fc gamma RII, but not anti-Fc gamma RIII. Myeloid cell lines HL-60 and U937 were unable to perform cytotoxicity without prior culture with rIFN-gamma, following which they killed CE through Fc gamma RI and Fc gamma RII, but were still incapable of HC lysis. THP-1, another myeloid cell line, was cytotoxic to CE through Fc gamma RI and Fc gamma RII without activation. Following rIFN-gamma treatment, cytotoxicity through these two Fc gamma R increased and was also mediated by Fc gamma RIII but these cells were still unable to kill HC.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Functional comparison of the inductions of NADPH oxidase activity and Fc gamma RI in IFN gamma-treated U937 cells.

The capacity to generate superoxide anion (O2-) can be induced in U937 cells by various agents known to cause myeloid cell differentiation. Other reported differentiation events include diminished cell proliferation and the induction by gamma-interferon (IFN gamma) of Fc receptors for immunoglobulin G1 (Fc gamma RI). In this study, we differentiated U937 cells and high Fc gamma RI-expression mutants of U937 cells by treating them with IFN gamma. We compared the time courses over which surface Fc gamma RI became maximal, NADPH oxidase activity was induced, and the antiproliferative effect of IFN gamma was detected. Oxidase activity was measured by stimulating cells with PMA or by activating surface Fc gamma RI using aggregated human IgG1 or second antibody crosslinking of mAb 32/Fc gamma RI complexes. We found that IFN gamma in the absence of additional lymphokines induced high levels of oxidase activity in maximally differentiated U937 cells with even higher levels in the fully differentiated high-Fc gamma RI expression mutants (greater than 8 nmoles/10(6) cells/min for A12.13 cells). Over the course of differentiation, maximal induced levels of Fc gamma RI were reached after 1 to 2 days of IFN gamma treatment, prior to the antiproliferative effect of the lymphokine. In contrast, oxidase activity was induced after a lag of approximately 2 days, becoming maximal only after 4 to 6 days of IFN gamma treatment. This comparison of the induction of Fc gamma RI with that of oxidase activity triggered through Fc gamma RI indicated that the rapid increase of surface receptor was not accompanied by a completion of the pathway of Fc gamma RI-mediated oxidase activity. However, the time courses of induction detected by PMA and Fc gamma RI-agonists were coincident suggesting that the development of oxidative capacity could be due to the induction of components required by both the PMA- and surface receptor-mediated pathways. There are several oxidase components that are known to be IFN gamma-inducible, such as the oxidase flavoprotein, a b558 cytochrome peptide, and oxidase-requiring cytosolic components, and it is possible that one or a set of these components could be the limiting factor(s) for IFN gamma-induced oxidase activity.

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.

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.

The effect of cytokines on the expression and function of Fc receptors for IgG on human myeloid cells.

We examined expression and cytotoxic triggering capability of the three Fc receptors for IgG (Fc gamma R) on human monocytes, PMNs and myeloid cell lines after in vitro culture with various cytokines. Fc gamma R expression was evaluated using specific anti-Fc gamma R monoclonal antibodies (mAb). The cytotoxic capability of each Fc gamma R was examined after the effector cells were treated with the recombinant cytokines IFN-gamma. TNF alpha, GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, or IL-6. Hybridoma cell lines (HC) bearing antibody directed to Fc gamma RI (HC 32), Fc gamma RII (HC IV.3) or Fc gamma RIII (HC 3G8) were used as targets, as were chicken erythrocytes (CE) sensitized with heteroantibodies composed of anti-Fc gamma R mAbs (32, IV.3, 3G8) linked to anti-CE antibody. Only IFN-gamma treatment significantly increased Fc gamma R expression and then only Fc gamma RI. IFN-gamma dramatically up-regulated Fc gamma RI expression on all cells tested. However, ADCC was enhanced by treatment with a number of cytokines other than IFN-gamma. GM-CSF, TNF, and IFN-gamma treatment enhanced killing of HC 32 and HC IV.3 by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through Fc gamma RII, whereas PMN killing of HC through Fc gamma RIII could not be induced by any of the cytokines studied. Although only IFN-gamma treatment increased ADCC of CE by monocytes, GM-CSF treatment as well as IFN-gamma treatment augmented ADCC of CE by PMNs. In addition to IFN-gamma treatment, IL-6 treatment enabled U937 cells to lyse CE. Whereas IFN-gamma-treated U937 cells killed CE through both Fc gamma RI and Fc gamma RII, IL-6-treated U937 cells killed CE only through Fc gamma RI. In addition to IFN-gamma treatment, G-CSF treatment enabled HL-60 cells to lyse CE through both Fc gamma RI and Fc gamma RII. These results demonstrate that although IFN-gamma appears unique in regulating Fc gamma R expression on myeloid cells, cytokines other than IFN-gamma affect ADCC by these cells in a receptor-specific manner.

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.

The effect of recombinant human interleukin-3 and recombinant human granulocyte-macrophage colony-stimulating factor on Fc gamma receptor expression and antibody-dependent cellular cytotoxicity of hematopoietic progenitor cells during in vitro myeloid maturation.

Enriched progenitor cell fractions from human bone marrow were induced to undergo myeloid maturation in culture using recombinant human interleukin-3 (rhIL-3) and granulocyte-macrophage colony stimulating factor (rhGM-CSF). A negative selection method using the murine monoclonal antibodies (MABs) PM81 (anti-CD15), AML-2-23 (anti-CD14), PC251 (anti-CD33), OKT11 (anti-CD2), and SCCL-1 (anti-CD71) and immunomagnetic beads coated with sheep anti-mouse IgG (Dynal A.S., Oslo, Norway) was used to remove the more mature cellular components of mononuclear cells from normal donor bone marrow samples. The resulting fraction of cells contained 35 to 40% CD34-positive cells, and less than 1% of cells expressed the receptors for the constant portion of immunoglobulin G Fc gamma RI or Fc gamma RII. A small population (3-5%) expressed Fc gamma RIII on day 0, and these cells were found by two-color flow cytometry to be primarily natural killer (NK) cells. The level of Fc gamma R expression was determined every 2 to 3 days on aliquots of the differentiating cells. Thirteen percent of the cultured bone marrow cells expressed Fc gamma RII after 48 hours in liquid culture with rhIL-3 and rhGM-CSF. The percent of cells expressing Fc gamma RII increased to a peak of 78% of the gated population on day 10. The mean fluorescence intensity (MFI) remained low for the first 8 to 10 days of culture, but at that time the MFI more than doubled. Fc gamma RI and Fc gamma RIII expression remained low throughout the culture period. The ability of the differentiating cells to perform antibody-dependent cellular cytotoxicity (ADCC) was determined at a single-cell level in a modified plaque assay using monolayers of ox erythrocyte (oxE) target cells. The purified progenitor cells, when placed in oxE monolayers sensitized with polyclonal rabbit anti-oxE antibody (AB), showed no plaque formation over control oxE layers. No increase in ability to generate cytolytic plaques in antibody-sensitized oxE layers was seen compared with unsensitized oxE layers until after 10 days of incubation in liquid culture. At that time, the percent of cells forming plaques in the AB-sensitized oxE layers was 34.4 +/- 10.7% (average +/- standard error of the mean [SEM]; n = 4) compared with 10.0 +/- 0.7% on the control oxE layers. The peak plaque formation appeared to coincide with the increase in MFI of a large population of the cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)