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The low-affinity Fc receptor for immune-complexed IgG (Fc gamma RIII; CD16) present on in vitro cultured human monocytes are encoded by an Fc gamma RIII-2 gene that, by cDNA sequence analysis, is identical to that expressed on tissue macrophages and on natural killer cells. In macrophages, Fc gamma RIII-2 encodes a glycoprotein of 52-62 kDa, with a peptide backbone of 33 kDa identical to that of the homologous receptor on natural killer cells. Like this and unlike in polymorphonuclear neutrophils, Fc gamma RIII (CD16) on cultured monocytes is insensitive to phosphatidylinositol-specific phospholipase C, is not allelic for the neutrophil NA alloantigens NA-1/NA-2, is not recognized by a monoclonal antibody (1D3) detecting an epitope present only on neutrophil Fc gamma RIII (CD16) and functions to trigger cytotoxicity upon ligand binding.

We report evidence that murine NK cells express a functional Fc gamma RII encoded by the Fc gamma RII alpha gene. Several lines of indirect evidence indicate that freshly obtained NK cells from mice of several strains bear a functional Fc gamma RII: (a) anti-Fc gamma RII antibody 2.4G2 detects a small but significant proportion of sIg- cells and a small proportion of the 2.4G2+ cells are included in the Thy-1+ population; (b) sIg- lymphocytes contain 2.4G2+ and Fc gamma R-bearing cells in similar proportions; (c) binding of particulate immune complexes by sIg- lymphocytes is completely inhibited by 2.4G2; (d) 2.4G2+ cells mediate greater than 50% of the spontaneous cytotoxicity in sIg- splenic lymphocytes. Direct evidence for the presence of Fc gamma RII on murine NK cells is provided by the results of two-color immunofluorescence studies performed on splenic lymphocytes from C57BL/6 mice showing coexpression of NK-1.1 and 2.4G2. Studies of in vitro propagated homogeneous NK cell populations confirm that murine NK cells express only Fc gamma RII and that this Fc gamma R is functional, as shown in experiments of inhibition of ADCC by the anti-Fc gamma RII antibody 2.4G2. The results of studies at the molecular level show that an Fc gamma RII alpha transcript identical to that expressed in macrophages is the only molecule encoding Fc gamma RII in murine NK cells.

We report evidence that FcR(CD16) on human NK cells are signal-transducing molecules that, upon ligand binding, induce transcription of genes encoding surface activation molecules [IL-2-R(CD25)] and cytokines (IFN-gamma and TNF) relevant to NK cell biology and functions. Homogeneous NK and T cell populations purified from short-term bulk cultures of PBMC with irradiated B lymphoblastoid cell lines were cultured in the presence of FcR ligands (particulate immune complexes or immobilized anti-CD16 antibodies) alone or with rIL-2. Upon 18 h of stimulation, NK cells express Tac, TfR, and 4F2 antigens and produce IFN-gamma and TNF; both effects are synergistically enhanced in the presence of rIL-2, which is itself ineffective. Treatment of NK cells with FcR(CD16) ligands induces accumulation of mRNA for IFN-gamma and TNF and, to a lesser extent, IL-2-R with fast kinetics also in the absence of de novo protein synthesis. rIL-2 and FcR(CD16) ligands synergize to induce mRNA accumulation. mRNA accumulation and transcription of TNF and IFN-gamma genes induced by FcR(CD16) ligands are greater than those induced by rIL-2, and the reverse is true for IL-2-R. The two stimuli do not synergize at the transcriptional level. These observations indicate that the mechanisms through which FcR(CD16) ligands and rIL-2 induce NK cell activation are, in part, distinct. Both operate at the transcriptional level, although other mechanisms are probably induced by the FcR ligand stimulus per se or in combination with other lymphokines and synergize at a post-transcriptional or translational level to enhance NK cell activation.

Phorbol esters with tumor promoter activity enhance the spontaneous cytotoxicity of human lymphocytes against a variety of target cell lines, with an efficiency that correlates with their potency as tumor promoters or skin irritants. Analysis of surface marker expression of the lymphocytes cytotoxic after treatment with phorbol ester identified the cytotoxic cell subset as that containing natural killer cells. Although gamma-interferon (IFN gamma) is produced by T cells treated with phorbol esters, IFN gamma is probably not the mediator of enhancement of natural killer cell activity, because anti-IFN gamma antibodies failed to block this enhancement. Spontaneous cell-mediated cytotoxicity is inhibited when phorbol esters are present during the cytotoxic assay, but is enhanced when the effector cells are pretreated with these agents. On the other hand, antibody-dependent cytotoxicity mediated by lymphocytes is inhibited by phorbol ester pretreatment of the effector cells or by phorbol esters present during the cytotoxic assay. Treatment of lymphocytes with phorbol esters at 37 degrees C, but not at 4 degrees C, completely abrogates in 1 to 2 hr the expression of the receptor for the Fc fragment of IgG, as detected by rosette formation with IgG-sensitized erythrocytes and by reactivity with anti-Fc receptor antibodies. The inhibition of antibody-dependent cytotoxicity by phorbol esters is probably secondary to their effect on the Fc receptor.

We compare five monoclonal antibodies ( B73 .1, 3G8 , Leu- 11a , Leu- 11b , and VEP13 ) that react with natural killer (NK) cells and polymorphonuclear cells (PMN). We show that all of these antibodies are directed against and inhibit the functional properties of the receptor for the Fc portion of IgG (FcR). Modulation of the FcR on NK cells after reaction with immune complexes induces the disappearance of the antigen(s) recognized by each of the five antibodies. Conversely, the antibodies block binding of IgG-sensitized erythrocytes to the NK cells and PMN and inhibit their ability to mediate cytotoxicity against antibody-sensitized tumor target cells. By using two-color immunofluorescence techniques, we characterize directly the lymphocyte population recognized by these antibodies and show that it is a homogeneous subset that does not bear markers of either B or T cells, with the exception of the 33,000 dalton antigen characteristic of suppressor/cytotoxic T cells present in 20 to 50% of the cells, and the 45,000 dalton receptor for sheep erythrocytes present on 80 to 90% of the cells. The phenotype of the cells reacting with the monoclonal antibodies corresponds to that of NK cells. Cross-competition experiments indicate that these antibodies detect at least two distinct epitopes on FcR, one ( B73 .1) preferentially expressed on NK cells and one or more ( 3G8 /Leu- 11a /Leu- 11b / VEP13 ) preferentially expressed on PMN. The lack of reactivity of these antibodies with B cells suggests that human B cells bear a different FcR from that on NK cells and PMN.

We describe the production of the monoclonal antibody B73.1, reacting with a subset of human lymphocytes and, in about one-half of the donors, with neutrophilic polymorphonuclear leukocytes. In the peripheral blood from normal adult donors, 14.6 +/- 8.5% of the lymphocytes react with B73.1 antibody. The B73.1(+) lymphocyte subset does not bear markers of typical T or B cells and corresponds to the lymphocyte subset containing antibody-dependent killer (K) and natural killer (NK) cells. We demonstrate that: a) virtually all lymphocytes with K/NK cytotoxic activity are found in the lymphocyte subpopulation bearing the B73.1-defined antigen; b) the B73.1(+) lymphocyte subset bears the combination of antigens known to be present on K/NK cells; and c) there is a positive correlation between the level of cytotoxicity and the actual number of B73.1(+) lymphocytes in individual donors. We also report the distribution of B73.1(+) lymphocytes according to donor age and tissue types. The use of the B73.1 antibody in quantitating the actual number of K/NK cells and in performing functional studies on spontaneous cytotoxicity is discussed.

Antibody 3G8 reacts with the receptor for the Fc fragment of aggregated IgG present on the majority of neutrophilic granulocytes and on a small proportion of lymphocytes. In this report, we compare the pattern of reactivity of antibody 3G8 on peripheral blood lymphocytes (PBL) and on polymorphonuclear leukocytes (PMN) with that of antibody B73.1, which reacts with the Fc receptor of natural killer (NK) cells or with a molecule functionally associated with it. We show that 3G8 reacts with the same PBL subset detected by antibody B73.1 and is responsible for virtually all NK cytotoxic activity. The lymphocyte subset recognized by the two antibodies has the morphology of large granular lymphocytes and includes neither B nor T cells. Our results indicate that NK cells and PMN express the same Fc receptor for immune complexes, and that B73.1 and 3G8 recognize on the same receptor two distinct epitopes that are preferentially expressed on NK cells and on PMN, respectively.

A low affinity receptor for IgG immune complexes, Fc gamma RIII(CD16), is expressed on human NK cells as an integral membrane glycoprotein anchored through a transmembrane peptide; on polymorphonuclear neutrophils (PMN) the receptor is anchored through a phosphatidylinositol (PI) linkage. The protein on NK cells has a molecular mass 6-10 kD larger than that on PMN, and, unlike the latter, is resistant to PI-specific phospholipase C (PI-PLC). Fc gamma RIII(CD16) transcripts isolated from PMN and NK cells of single donors revealed multiple single nucleotide differences, one of which converts an in frame UGA termination codon to a CGA codon. The resulting open reading frame encodes a longer cytoplasmic domain for Fc gamma RIII(CD16) in NK cells, contributing to its transmembrane anchor. Two nearly identical, linked genes that encode these transcripts have been cloned for Fc gamma RIII(CD16), one of which (III-1) is allelic for NA-1 and NA-2. The allelic sites have been mapped to two single nucleotides in the extracellular domain. These genes are transcribed in a cell type-specific fashion to generate the alternatively anchored forms of this receptor.

The role of IgG antibody in the sensitization of human basophils and mast cells to antigen is uncertain. To help resolve this uncertainty, we characterized by two-color fluorometric analysis the Fc receptors for IgG (Fc gamma R) on human basophils. Basophil-containing mononuclear cell fractions of atopic and nonatopic adult volunteers were incubated sequentially with fluorescein isothiocyanate-conjugated murine monoclonal IgE and biotinylated monoclonal antibodies (MAb) that bind specifically to the different Fc gamma R subtypes. Binding of biotinylated MAbs was visualized after subsequent incubation with phycoerythrin-strepavidin conjugate. Basophils did not react with a murine monomeric IgG2a, which binds specifically through its Fc to the high-affinity Fc gamma RI (CD64), or with MAbs specific for the low-affinity Fc gamma RIII (CD16). However, basophils reacted with a MAb specific for the low-affinity Fc gamma RII (CDw32). The profile of basophil Fc gamma R expression was not altered after brief IgE-mediated activation. In addition, pretreatment with gamma interferon, which induced expression of Fc gamma RI (CD64) on neutrophils, did not induce Fc gamma RI expression on basophils. These results indicate that the Fc gamma R present on basophils is exclusively of the Fc gamma RII (CDw32) subtype. The absence of the high-affinity Fc gamma RI (CD64) suggests that antigenic sensitization of basophils by monomeric IgG does not occur.

In this study, we present evidence that interaction of Fc gamma R(CD16) with ligands (immune complexes or anti-CD16 antibodies) induces a rapid rise in [Ca2+]i and fast production of both inositol 1,4,5 triphosphate (IP3) and IP4 in homogeneous NK cell preparations. Part of the initial [Ca2+]i rise observed upon stimulation of NK cells with either anti-CD16 antibodies alone or after their crosslinking at the cell membrane depends on Ca2+ mobilization from intracellular stores, but sustained [Ca2+]i levels are maintained, after the initial spike, through influx of extracellular Ca2+. The [Ca2+]i rise is mediated, at least in part, by increases in IP3 after receptor-induced hydrolysis of membrane polyphosphoinositides (PPI). The role of extracellular Ca2+ in Fc gamma R(CD16)-dependent induction of lymphokine gene expression has been tested by evaluating production, mRNA accumulation and transcription of IFN-gamma and TNF in NK cells stimulated with Fc gamma R(CD16) ligands and/or rIL-2 in the presence of EGTA. Under these conditions, accumulation and transcription of both IFN-gamma and TNF mRNA induced by CD16 ligands, but not that induced by rIL-2, is completely abolished and neither cytokine can be detected at significant levels in the supernatant fluids of cells so treated. These data confirm that NK cell activation by specific ligands occurs through mechanisms distinct from those induced by IL-2, and indicate that extracellular Ca2+ represents a stringent requirement for cytokine production induced in NK cells through specific (Fc gamma R) stimulation. Our data also indicate that the [Ca2+]i rise induced upon Fc gamma R(CD16) crosslinking, though necessary, is not sufficient per se to induce activation of lymphokine genes, compatible with the hypothesis that Fc gamma R(CD16) crosslinking generates additional transducing signals that synergize with IL-2 to maximally activate NK cells.

We report here that FcR for human monomeric IgG1 can be induced on cells of myeloid origin cultured in the presence of IFN gamma for 8 h. Supernatant fluids from cultures of lymphocytes infected with a variety of viruses or cocultured with cell lines have the same FcR enhancing effect as IFN gamma. We identify the factor in the supernatant fluid responsible for the induction as immune interferon. Among the different types of IFN, only the gamma type (both purified and recombinant) specifically induces the appearance of FcR for monomeric IgG1 on normal and leukemic myeloid cells but not on cells of lymphoid origin. This effect is also evident on mature PMN. We show that the specificity and the affinity of the receptor induced on HL-60 promyelocytic cells, peripheral blood monocytes, and PMN are identical to those of the receptor spontaneously present on the same cells, except for PMN, which do not spontaneously express this type of receptor. The results of inhibition experiments performed with mouse IgG of and IgG3. These results suggest that the receptor present on human monocytes different isotypes indicate that the receptor can be inhibited by murine IgG2a or immature myeloid cells, selectively inducible by IFN gamma, has a specificity similar to the FcR1 described on mouse macrophages.

We report here a comparative study of the effects of several cytokines known to affect myeloid cell differentiation on functional properties of human mature granulocytes. We show that recombinant interferon-gamma (rIFN-gamma), recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF), recombinant tumor necrosis factor (rTNF), and lymphotoxin (LT) purified to homogeneity are potent stimulators of polymorphonuclear cells (PMN) activity. All cytokines enhance antibody-dependent cell-mediated cytotoxicity (Ab-CMC) mediated by human PMN; however, rGM-CSF, rTNF, and LT have an immediate and short-lived effect on the PMN, whereas the activation by rIFN-gamma requires several hours of induction but can be observed up to 24 to 48 hr of culture. Only the effect of rIFN-gamma is in part dependent on induction of a high-affinity FcR for monomeric IgG on PMN, as suggested by two-color sorting analysis, and on mechanisms that result in prolonged survival of PMN in a functionally active state to mediate oxidative burst, phagocytosis, and bactericidal activity. Greater enhancement of Ab-CMC is obtained by using rIFN-gamma in combination with the other cytokines. Our data indicate that cytokines previously defined on the basis of their cytotoxic effects mediate a wide spectrum of activities on mature myeloid cells and provide evidence for their possible role in vivo, alone or in combination with rIFN-gamma, in modulating functional activities of cells responsible for non-adaptive systems of defense.