FC gamma receptor mediated phagocytosis by human neutrophil cytoplasts.

Neutrophils utilize Fcgamma Receptors (FcgammaR) to bind and internalize antibody coated cells or immune complexes. We have compared ultrastructurally FcgammaR-mediated phagocytosis by neutrophil cytoplasts (enucleated and granule-free neutrophils) prepared either by treatment with cytochalasin B (CB-cytoplasts) followed by ultracentrifugation or by brief heating at 45 degrees C in suspension followed by ultracentrifugation. The phagocytosis of antibody-coated erythrocytes (EIgG) or the internalization of crosslinked IgG complexes by cytoplasts prepared by brief heating was comparable to that of untreated neutrophils. In contrast, CB-cytoplasts bound but failed to internalize ElgG or crosslinked IgG complexes. Comparison of these two methods for the preparation of neutrophil cytoplasts may assist in clarifying the signal transduction requirements involved following ligand binding to FcgammaR which initiate phagocytosis.

Antineutrophil cytoplasmic antibodies preferentially engage Fc gammaRIIIb on human neutrophils.

Antineutrophil cytoplasmic Abs (ANCA) are found in the circulation of many patients with systemic vasculitis. ANCA bind to ANCA target, such as proteinase 3 and myeloperoxidase, and activate neutrophils in an Fc gammaR-dependent manner. Human neutrophils constitutively express Fc gammaRIIa (CD32) and Fc gammaRIIIb (CD16), and there is clear in vitro experimental evidence of ANCA-mediated engagement of Fc gammaRIIa. However, direct experimental evidence of ANCA engagement of neutrophil Fc gammaRIIIb has been obscured by technical problems related to activation-induced receptor shedding and activation-induced expression of receptor on the surface of neutrophils. In this study, by blocking receptor shedding and using appropriate reporter anti-Fc gammaR mAb, we show that human cANCA and pANCA, and murine mAb with corresponding reactivities, can indeed engage Fc gammaRIIIb. Furthermore, our data suggest that Fc gammaRIIIb is preferentially engaged by ANCA relative to Fc gammaRIIa presumably due to the nearly 10-fold excess of Fc gammaRIIIb expression relative to Fc gammaRIIa expression. These results clearly demonstrate that the Fc region of ANCA bound to an ANCA target on the neutrophil surface engage Fc gammaRIIIb and indicate that Fc gammaRIIIb and Fc gammaRIIa may both be active participants in ANCA-induced neutrophil activation. However, given the low levels of ANCA target expression on neutrophils from patients with systemic vasculitis, Fc gammaRIIIb is likely to play a critical role in initiating and perpetuating ANCA-induced neutrophil activation.

Recombinant human interleukin-3 enhances the in vitro survival of mononuclear phagocytes from the peripheral blood of very low birth weight premature infants at birth.

Interleukin 3 (IL-3) is a pluripotent hematopoietic growth factor that stimulates proliferation, differentiation, and function of multiple cell lineages. We examined the effects of recombinant human IL-3 (rhIL-3) on peripheral blood mononuclear cells (MNC) isolated from 18 premature human newborns with birth weights between 600 and 1,500 g at birth and at 2 and 4 weeks of age, from 7 full-term neonates, and from 26 normal adult volunteers. After 2 weeks in liquid culture, rhIL-3 treatment was associated with a six- to nine-fold increase in the survival of MNC from very low birth weight (VLBW) neonates. In the absence of rhIL-3, VLBW neonatal MNC exhibited a low survival rate. MNC from 6 of 7 full-term neonates responded similarly to rhIL-3 with an eight-fold increase in survival. In contrast, rhIL-3 showed only a 20-30% increase in the survival of adult MNC (p = NS). When analyzed by immunofluorescent microscopy using monoclonal antibodies to phenotypic markers characteristic of individual MNC lineages, 70-80% of the surviving VLBW neonatal MNC were mononuclear phagocytes, while 70-80% of the surviving adult MNC were T cells. Full-term MNC cultures displayed a population of cells with an intermediate phenotype. Following rhIL-3 treatment, surviving MNC from term neonates displayed 35% T cells and 53% mononuclear phagocytes which was not significantly different from untreated MNC. rhIL-3 treatment was associated with a seven- to twelve-fold increase in the number of progenitor cells (CD34+) from VLBW neonatal blood and a three- to five-fold increase in the number of adult progenitor cells. Full-term neonates had a lower percentage of CD34+ cells than VLBW neonates, and this was not significantly altered by the rhIL-3 treatment. We conclude that rhIL-3 increases the number of mononuclear phagocytes that survive in culture following isolation from the peripheral blood of VLBW neonates. These in vitro studies may have a predictive value for in vivo studies utilizing combinations of hematopoietic growth factors to enhance neonatal host defense.

Extremely low birth weight infants have lower Fc gamma RIII (CD 16) plasma levels and their PMN produce less Fc gamma RIII compared to adults.

The purpose of this study was to determine whether decreased Fc gamma RIII expression on the PMN of extremely low birth weight infants (ELBW) is due to decreased receptor synthesis or increased receptor shedding from the PMN surface. 42 ELBW, 12 larger infants and 14 adults were enrolled. Plasma and total cellular Fc gamma RIII were measured by ELISA, and PMN Fc gamma RIII expression was measured by flow cytometry. ELBW PMN plasma membrane expression of Fc gamma RIII as measured by log mean channel fluorescence (5.00 +/- 1.98 vs. 10.68 +/- 1.61, p < 0.050) and plasma Fc gamma RIII levels were both lower (7.5 +/- 6.1 vs. 82.4 +/- 64.8 nM, p < 0.05) than in adult controls. In follow-up studies, 14 ELBW (age = 29 +/- 14 days, range = 14-56 days) increased PMN expression of Fc gamma RIII (p < 0.001) but not plasma Fc gamma RIII. ELBW had lower total PMN-associated Fc gamma RIII than adults (2.3 +/- 0.9 vs. 6.8 +/- 2.2 ng/10(6) PMN, p = 0.006). ELBW's PMN produce less Fc gamma RIII than adults' PMN, and expression of this receptor is developmentally regulated.

Tyrosine phosphorylation and association of Syk with Fc gamma RII in monocytic THP-1 cells.

Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor.

Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells.

Aggregation of Fc gamma RII (CD 32), a low affinity receptor for immunoglobulin G (IgG), on the monocytic cell line THP-1 induces protein tyrosine kinase (PTK) activity. Several distinct cellular proteins, including Fc gamma RII itself, are phosphorylated on tyrosine following cross-linking of the receptor. Fc gamma RII lacks intrinsic PTK activity. In this report we demonstrate that a kinase activity was coprecipitated with Fc gamma RII in THP-1 cells. The kinetics of the receptor-associated kinase activity paralleled the appearance of tyrosine phosphorylation events observed following Fc gamma RII activation of THP-1 cells. Several proteins were associated with the receptor. Reimmunoprecipitation analysis demonstrated that lyn gene products were among the proteins coprecipitated with Fc gamma RII. p59hck (Hck) and p56lyn (Lyn) were the most abundant Src-related PTKs (Src-PTKs) in THP-1 cells. Enzymatic activity of both kinases, as measured by an in vitro kinase assay, was increased following specific cross-linking of Fc gamma RII. Furthermore, Fc gamma RII was specifically associated with both enzymes following its engagement and served as a substrate for both of these kinases. The association of Fc gamma RII with Src-PTK was specific for Fc gamma RII activation of THP-1 cells, since activation of cells via the high affinity Fc gamma receptor, Fc gamma RI (CD 64), did not result in association of Fc gamma RII with Hck or Lyn. Our data demonstrate a functional and physical association of Fc gamma RII with Hck and Lyn consistent with the involvement of Src-PTK in Fc gamma RII-mediated signal transduction.

Tyrosine phosphorylation provides an obligatory early signal for Fc gamma RII-mediated endocytosis in the monocytic cell line THP-1.

The human monocytic cell line THP-1 expresses two classes of IgG Fc receptor (Fc gamma R), Fc gamma RI, a high affinity 72-kDa Fc gamma R, and Fc gamma RII, a low affinity 40-kDa Fc gamma R. Biochemical as well as indirect immunofluorescence studies demonstrated that the selective cross-linking of Fc gamma RII with either anti-Fc gamma RII mAb Fab followed by F(ab)2 fragments of goat anti-mouse IgG, or aggregated hIgG1, which represents a physiologic ligand for this receptor, resulted in the activation of a protein tyrosine kinase (PTK). Several distinct cellular proteins including the Fc gamma RII itself were specifically phosphorylated on tyrosine upon ligand binding. Cross-linking of Fc gamma RII also triggered a rapid internalization of Fc gamma RII that was dependent upon tyrosine kinase activity. The internalization of the receptor in endocytic vesicles was established by confocal microscopy. The time course of Fc gamma RII-initiated tyrosine phosphorylation paralleled endocytic events and reached a maximum between 5 and 10 min after ligand binding and declined toward basal levels as endocytosis was completed. Identical concentrations of genistein, an inhibitor of PTK, blocked Fc gamma RII-mediated endocytosis as well as the induction of tyrosine phosphorylation of Fc gamma RII and other cellular proteins. Cross-linking of Fc gamma RI also induced a rapid tyrosine phosphorylation of cellular proteins similar to the Fc gamma RII-mediated events. However, Fc gamma RII was not tyrosyl phosphorylated upon Fc gamma RI activation. Thus Fc gamma RII is a unique substrate for the PTK activity associated with Fc gamma RII upon cross-linking of this receptor. These results support the conclusion that Fc gamma RII is capable of independent signaling on monocytic cells and that protein tyrosine phosphorylation is an obligatory proximal signal for Fc gamma RII-mediated endocytosis. Furthermore, the signaling pathways employed by Fc gamma RI and Fc gamma RII are likely to be distinct.

Identification of Fc gamma RI, II and III on normal human brain ramified microglia and on microglia in senile plaques in Alzheimer's disease.

Using monoclonal antibodies to the three known human leukocyte IgG receptors, Fc gamma R, we examined the expression of Fc gamma R in normal brains and in Alzheimer's disease. We found Fc gamma RI, II and III immunoreactivity in senile plaques and on ramified microglia throughout the cortex and white matter of normal and Alzheimer's disease brains. Fc gamma RI expression was independently confirmed by a murine isotype binding study. These findings suggest that intrinsic Fc gamma R may play an important role in normal and disordered immune-related processes in the brain. They support the idea that microglia are brain macrophages.

Poliovirus receptor on human blood cells: a possible extraneural site of poliovirus replication.

In order to determine whether any primary human blood cells have the ability to replicate poliovirus (PV), peripheral blood cell components were isolated and analyzed for their cell surface expression of the poliovirus receptor (PVR). Following two-color immunfluoresence staining with lineage-specific markers, the cells were analyzed by flow-cytometric methods. PVR cell surface expression was detected on most mononuclear cells expressing CD14, a marker for mononuclear phagocytes. There was no PVR cell surface expression on platelets and extremely low levels on polymorphonuclear leukocytes. Mononuclear leukocytes from Ficoll density centrifugation were found to support PV replication. The finding of PVR on mononuclear phagocytes and the ability of primary human blood cells to support PV replication in the absence of cultivation has implications for both the normal and pathogenic role of PVR.

Soluble Fc gamma RIII is present in lower concentrations in the serum of patients with acute myelogenous leukemia (AML): a retrospective study.

Fc gamma RIII is a low affinity immunoglobulin G receptor expressed by neutrophils, natural killer cells, and macrophages. Soluble forms of Fc gamma RIII have been identified in serum, plasma, and other body fluids. Previous studies showed that Fc gamma RIII appeared late in myeloid differentiation. This retrospective study was designed to measure the concentration of soluble Fc gamma RIII in serum from patients with acute myelogenous leukemia (AML), a disease generally characterized by granulocytopenia and an increase in circulating myeloblasts and occasionally promyelocytes. Frozen serum samples from patients with AML and from age-matched normal donors were obtained from the Biological Carcinogenesis Branch Repository of the National Cancer Institute. We used an ELISA to measure the concentration of soluble Fc gamma RIII in these serum samples and observed significantly lower concentrations of soluble Fc gamma RIII in the serum of AML patients. The mean concentration of soluble Fc gamma RIII was 9.5 nM in normals (n = 48) and 5.4 nM in AML patients (n = 46), (p < 0.0005). Whether this difference is due to defects in granulopoiesis in these patients or to other parameters of the disease is unknown at this time. Our retrospective study should provide the basis for subsequent investigation of patients with AML to correlate soluble Fc gamma RIII concentrations with the clinical status of the patients.

Differential roles of Fc gamma RII and Fc gamma RIII in immune complex stimulation of human neutrophils.

Insoluble immune complexes (IIC) stimulate human neutrophils through Fc gamma receptors. Freshly isolated human neutrophils express two FcR subclasses, FcRII and FcRIII. We explored the role of FcRII and FcRIII in this activation process by selectively binding each FcR subclass with the Fab fragments of the respective anti-FcR monoclonal antibodies (MFab) before exposure to IIC. Correlation among liganded FcR subclass, IIC binding, and ensuant IIC stimulation was achieved with multiparameter flow cytometry. We utilized rhodamine-labeled anti-FcRIII and fluorescein-labeled IIC to study binding and observed the change in [Ca2+]i in the same cell with a Ca2+ indicator, Indo-1. Treatment with either anti-FcRII (IV.3) or anti-FcRIII (3G8) MFab decreased both the fraction of cells exhibiting a Ca2+ transient and the magnitude of that transient, although only anti-FcRIII but not anti-FcRII significantly inhibited the subsequent IIC binding. In addition, cells treated with anti-FcRII and then stimulated with IIC exhibited a decrease in both the intracellular Ca2+ transient and the later Ca2+ influx, whereas anti-FcRIII totally abolished the mobilization of intracellular Ca2+ without affecting the Ca2+ influx. Treatment with either anti-FcR MFab decreased the IIC-stimulated transmembrane potential change, oxidative burst, and elastase release. These studies indicate that freshly isolated neutrophils' Fc receptor subclasses have unique roles in the IIC-initiated stimulation and that full activation can only be achieved when both FcR subclasses are available.

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)

A soluble form of Fc gamma RIII is present in human serum and other body fluids and is elevated at sites of inflammation.

We have developed a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) to measure the concentration of Fc gamma RIII in serum and other body fluids. This ELISA is based on the use of monoclonal antibody (MoAb) (3G8) to Fc gamma RIII and a rabbit antiserum against Fc gamma RIII. The lower limit of detection of this ELISA was 1.5 nmol/L. The concentration of soluble Fc gamma RIII in normal serum ranged from 7.3 to 75.9 nmol/L. Soluble Fc gamma RIII was also present in other normal biologic fluids such as saliva, urine, and seminal fluid, but at much lower concentrations than that found in serum. Rabbit anti-Fc gamma RIII immunoblotted polypeptides immunoprecipitated with MoAb 3G8. Fc gamma RIII immunoprecipitated from a neutrophil lysate migrated from 40 to 76 Kd, whereas Fc gamma RIII immunoprecipitated from serum from the same donor migrated from 40 to 66 Kd. The soluble form of Fc gamma RIII apparently was bound to serum IgG, because immunoprecipitation of soluble Fc gamma RIII by MoAb 3G8 coprecipitated polypeptides that were identified by goat antihuman IgG. Incubation of neutrophils in vitro at 4 degrees C and 37 degrees C showed that Fc gamma RIII was released after 30 minutes of incubation at 37 degrees C. To determine whether there was a correlation between the concentration of soluble Fc gamma RIII in biologic fluids and inflammatory diseases, we measured the concentration of Fc gamma RIII in the bronchoalveolar lavage fluid from patients with adult respiratory distress syndrome (ARDS) and in the synovial fluid from patients with various forms of arthritis. In ARDS, we found concentrations of soluble Fc gamma RIII that were five to seven times higher than that found in the bronchoalveolar lavage fluids from healthy adults. The concentration of soluble Fc gamma RIII in the synovial fluid from patients with rheumatoid arthritis ranged from 10 nmol/L to 28 mumol/L. These results suggest that activated neutrophils, such as those at sites of inflammation, may release Fc gamma RIII.

Monoclonal antibodies identify Fc gamma receptors on unfertilized human oocytes but not spermatozoa.

Oolemmal Fc receptors have previously been shown to play a role in the promotion of adhesion by antibody labeled human spermatozoa to zona-free hamster eggs. In this work, we demonstrated the presence of Fc gamma RI, Fc gamma RII and Fc gamma RIII on the oolemma of unfertilized human oocytes by means of monoclonal antibodies directed against these receptors, detected both by immunobead rosetting and indirect immunofluorescence. These receptors were also functionally active in that they were able to bind human aggregated IgG, human IgG-Fc, mouse IgG1 and IgG2a. While the presence of oolemmal IgG-Fc receptors might play a role in reproductive failure, by their promotion of polyspermic fertilization, in cases where antisperm antibodies bound to the spermatozoan surface, their role in the normal physiology of fertilization or in other events unrelated to sperm incorporation remains to be determined. In contrast, Fc gamma receptors were not present on human spermatozoa, irrespective of their functional state (fresh ejaculated, capacitated or acrosome reacted).

Detection of tumor necrosis factor induced nuclear damage with p-phenylenediamine.

A simple technique has been used to observe the effects of rTNF alpha on nuclear morphology. Using the intercalating dye p-phenylenediamine to stain nuclei, we detected TNF alpha-induced nuclear alterations which characteristically occur during apoptosis in the TNF alpha sensitive U937 cell line. Nuclear alterations were visible prior to the loss of plasma membrane integrity and subsequent cell death. A subclone of U937 cells was isolated in which TNF alpha failed to alter either cell viability or nuclear morphology. TNF alpha resistant U937 cells, however, retained the ability to bind, internalize and degrade TNF alpha. These results suggest that nuclear damage induced by TNF alpha in sensitive U937 cells occurs early and precedes cell death as measured by dye exclusion assays. Staining cells with p-phenylenediamine and visualization with a 520 nm fluorescence filter provides a rapid and simple method to monitor apoptotic cell death.

A common epitope is recognized by monoclonal antibodies prepared against purified human neutrophil Fc gamma RIII (CD16).

Fc gamma RIII is one of two Fc gamma R constitutively expressed by human neutrophils. We have prepared a panel of anti-Fc gamma RIII mAb following immunization of mice with Fc gamma RIII purified from human neutrophils. Ten mAb which reacted with neutrophils, NK cells, and monocyte-derived macrophages were produced. Immunohistochemical staining demonstrated that these mAb also identified macrophages in the red pulp of spleen. Competitive cross-inhibition binding assays demonstrated that nine of the ten mAb reacted with a common epitope that is spatially associated with the ligand binding site. These nine mAb blocked the binding of immune complexes to neutrophils by 65 to 90%. In addition, two other anti-CD16 mAb, which also blocked immune complex binding to neutrophils, inhibited the binding of each of these nine mAb to neutrophils. One of the mAb produced here, 214.1, failed to block immune complex binding. In addition to immunoprecipitating the native Fc gamma RIII glycoprotein, mAb 214.1 was capable of immunoprecipitating a 28-kDa polypeptide following deglycosylation of Fc gamma RIII isolated from neutrophils. The results of cross-competition experiments suggest that mAb 214.1 may recognize the epitope identified by mAb BW209/2. Thus mAb 214.1 identifies a polypeptide epitope distinct from the ligand binding site of Fc gamma RIII on neutrophils.

Characterization of human alveolar macrophage Fc gamma receptor III: a transmembrane glycoprotein that is shed under in vitro culture conditions.

Three classes of Fc gamma receptors (FcR) have been identified on blood leukocytes: FcRI, FcRII, and FcRIII. Two forms of FcRIII have recently been characterized; a phosphatidylinositol linked form is found on neutrophils, whereas a transmembrane form of the molecule is found on a subset of peripheral blood lymphocytes. Peripheral blood monocytes express low levels of FcRIII on their surface, whereas FcRIII is readily expressed by tissue macrophages. The purpose of this investigation was to characterize the form of FcRIII expressed by normal human alveolar macrophages (AM) obtained from normal subjects by bronchoalveolar lavage. We found FcRIII expressed by AM has a molecular mass of 50 to 60 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis and migrates as a single band with a molecular mass of 35 kD after digestion with endoglycosidase F. Macrophage FcRIII was resistant to cleavage by phosphatidylinositol-specific phospholipase C. These results demonstrate that FcRIII expressed by AM is a transmembrane glycoprotein similar to the molecule found on peripheral blood lymphocytes. Scatchard binding analysis using 125I-labeled mAb 3G8 showed that AM express similar numbers of FcRIII as found on neutrophils (73,300 +/- 16,300 versus 69,300 +/- 8,500 receptor sites/cell, respectively; P = 0.73), whereas fewer binding sites were found on FcRIII-positive peripheral blood lymphocytes (35,300 +/- 13,900; P = 0.04). Of note, we found expression of FcRIII by AM was selectively and dramatically reduced during short term in vitro incubation at 37 degrees C. Receptor shedding as a result of proteolytic cleavage is probably responsible for the reduced expression that occurs during short-term in vitro culture.

Plasma membrane and intracellular pools of transferrin receptors decline during in vitro cultivation of U937 cells.

Transferrin receptor expression in the monocyte-like cell line U937 was investigated during in vitro cultivation. U937 cells expressed a single class of high affinity surface transferrin receptors (KD approximately 4 nM), with apparent subunit Mr of 90-95,000 Da as determined by SDS-reducing PAGE. [125I]-transferrin binding studies on detergent-solubilized cells revealed that half to two-thirds of the total functional binding sites were located intracellularly. Radioligand binding, immunofluorescence and flow cytometry studies were performed on intact, detergent-solubilized, or saponin-permeabilized cells, using either transferrin or the anti-transferrin receptor monoclonal antibody OKT9 IgG. These studies demonstrated that functional and antigenic transferrin receptor levels were maximal on cells 24 h after subculture at low density and declined during the culture period. Scatchard analysis of radioligand binding data suggested that the decline in functional transferrin binding sites resulted from a decline in the number of available receptors. These results demonstrate that in U937 cells there is a density-dependent regulation of transferrin receptor expression, resulting in a loss of functional and antigenic receptors from both plasma membrane and intracellular locations.

The human monocyte-like cell line THP-1 expresses Fc gamma RI and Fc gamma RII.

THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized Fc gamma R expression on this cell line by flow cytometry, radiolabeled IgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-Fc gamma RI, II, and III mAb, and a rabbit anti-Fc gamma RIII F(ab')2 demonstrated that only Fc gamma RI and Fc gamma RII are expressed by these cells. A panel of anti-Fc gamma RIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (Fc gamma RI) and of 42 to 53 kDa (Fc gamma RII). Fc gamma R expression was determined by binding of radioiodinated human IgG1 (to detect Fc gamma RI), mAb IV.3 (to detect Fc gamma RII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 x 10(-9) M) for IgG1 were found on THP-1 cells. Interferon-gamma (IFN gamma) upregulated Fc gamma RI expression by THP-1 cells 2.8-fold, whereas Fc gamma RI on U937 cells was increased six- to eight-fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor-alpha (TNF alpha), and vitamin D3 had no effect on IgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFN gamma treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D3 treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.3 identified 50,000 sites per cell. Treatment of THP-1 cells with IFN gamma, TNF alpha, PMA, or vitamin D3 had no effect on Fc gamma RII expression. That Fc gamma RI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human IgG1 as well as mouse IgG2a mAb to Fc gamma RII inhibited immune complex binding by 76 to 84%, whereas mouse IgG1 mAb to Fc gamma RII had minimal effect on immune complex binding. Fc gamma R expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D3 was able to induce the expression of CD14, a marker of mature monocytic phenotype.

Monoclonal antibodies to human neutrophil Fc gamma RIII (CD16) identify polypeptide epitopes.

Human neutrophils constitutively express two low-affinity Fc gamma R, Fc gamma RII (CD32) and Fc gamma RIII (CD16). Eleven monoclonal antibodies (mAb) to CD16 were used to identify antigenic differences among Fc gamma RIII-bearing cells, to define functional epitopes of Fc gamma RIII on neutrophils, and to characterize biochemically the epitopes identified by some of these mAb. Flow cytometry demonstrated that 9 of the 11 mAb reacted with neutrophils, 10 of the 11 reacted with natural killer cells, and 9 of 11 reacted with monocytes and monocyte-derived macrophages. These mAb reacted with CD16 positive cells with varying fluorescence intensities. The ability of anti-CD16 mAb to block the binding of 125I-labeled immune complexes to neutrophils was examined. Four monoclonal antibodies strongly inhibited (87-96%) the binding to neutrophils of 125I-labeled immune complexes. Competitive binding assays were performed to determine whether any other anti-CD16 mAb identify the epitope identified by mAb 3G8. Two other mAb, CLBFCGRAN 1 and CLBGRAN 11, blocked binding of 125I-3G8 IgG to neutrophils. Six of the anti-CD16 mAb efficiently immunoprecipitated polypeptides of broad mobility ranging from 45 to 84 kDa from 125I-labeled neutrophils. When Fc gamma RIII, a complex sialoglycoprotein consisting of almost 50% oligosaccharides, was immunoprecipitated from neutrophils with 3G8 Fab Sepharose and subsequently digested with N-glycanase, 5 of the 6 mAb were capable of immunoprecipitating a deglycosylated polypeptide migrating at 29 kDa. These results demonstrate that these 5 mAb identify polypeptide epitopes of Fc gamma RIII, whereas 1 mAb, YFC120.5, may react with a glycosyl moiety or a determinant whose conformation is dependent on the presence of oligosaccharides.