The effect of recombinant interferon-gamma on human monocyte-derived macrophages

The effect of recombinant interferon-gamma (rIFN-gamma) on human macrophage functions was studied, using monocytes which had matured to macrophages within hydrophobic containers. Following exposure to rIFN-gamma, the number of surface-expressed specific IgG-binding sites was increased. This increase was restricted to high-affinity Fc receptors (FcR), however; low-affinity FcR were not increased in number. Exposure to rIFN-gamma led to an enhanced chemiluminescence (CL) signal in the presence of luminol and a variety of respiratory burst stimuli, such as zymosan, phorbol 12-myristate 13-acetate or IgG-sensitized sheep erythrocytes (EA). In contrast, phagocytosis of EA was markedly depressed in rIFN-gamma-treated cells. Both increase in CL response and decrease in phagocytic activity were manifest after 1 day of treatment and were more pronounced after 2 days. While 5 U/ml of rIFN-gamma was an insufficient dose, 50 to 5000 U/ml yielded significant dose-dependent changes in both functional assays. Thus, using rIFN-gamma as a biological response-modifier, FcR expression and FcR-mediated CL can be dissociated from FcR-mediated phagocytosis.     

Monomeric and dimeric IgG1 as probes for assessing high-affinity and low-affinity receptors for IgG on human monocyte-derived macrophages and on activated macrophages

From a panel of IgG1 myeloma proteins, only one was found to interact with human monocyte FcR in a manner similar to that of polyclonal IgG. This protein was used in binding studies involving human macrophage Fc receptors. A monomeric fraction depleted of dimeric and polymeric IgG1 was crosslinked with bis-diazonium benzidine, and a fraction highly enriched in cross linked IgG1 dimers was radiolabeled. Labeled monomeric and dimeric IgG were allowed to interact with monocytes that had matured to macrophages in vitro. The association with macrophages at 4 degrees C, in the presence of cytochalasin B, reached a plateau after 6 hr. The dissociation induced by excess unlabeled IgG followed similar kinetics as the association, but 20-30% of the bound IgG could not be dissociated. Under equilibrium conditions, evidence for a single FcR population binding monomeric IgG was obtained, the Kd being in the range of 12-42 nM. In contrast, the binding of dimeric IgG was more consistent with a model assuming two populations of binding sites when appropriate curve-fitting calculations were applied. The high-affinity FcR population had a Kd in the range of 0.8-3.5 nM, whereas the Kd of the low-affinity FcR population was in the range of 28-85 nM. When macrophages had been pre-treated with recombinant interferon-gamma, the expression of high-affinity sites was increased by a factor of 1.5-3, but the number of low-affinity sites was not augmented. Cytofluorographic analyses confirmed the increased expression of high-affinity FcR, binding fluoresceinating murine IgG2a. The expression of CD16, a low-affinity FcR expressed on neutrophils, NK cells and macrophages, as well as the expression of the complement receptor type III was little influenced by the rIFN-gamma pretreatment.     

FcR-independent antibody-mediated cellular cytotoxicity

In this study we used palmitate-derivatized antibodies (pal-Ab) to examine the minimum contribution of Fc receptors (FcR) to macrophage (M phi)-mediated lysis and phagocytosis in antibody-dependent cellular cytotoxicity (ADCC). Pal-Ab specific for chicken erythrocytes (CE) were incorporated into the plasma membranes of M phi by insertion of the palmitate hydrocarbon chains into the outer leaflet of the phospholipid bilayer. In this system, the palmitate anchor bypassed the requirement for FcR in antibody-dependent effector-target conjugation and provided a unique opportunity to uncouple antibody-FcR interactions in ADCC. We show that binding of CE targets by P388D1 M phi effector cells through anti-CE pal-F(ab')2 can lead to efficient extracellular target cell lysis, but not phagocytosis. In contrast, pal-F(ab')2-mediated interactions between CE and peritoneal exudate M phi (PEM) activated both ingestion and extracellular lysis of the targets. In normal ADCC, FcR-dependent interactions between CE and either P388D1 cells or PEM triggered both extracellular lysis and phagocytosis. Our results demonstrate that lysis and phagocytosis in CE-directed ADCC by M phi have different minimum requirements for FcR functions. Moreover, our results suggest that FcR-independent triggers on the PEM surface are capable of triggering target cell lysis and internalization following antibody-mediated interactions.     

Photometric assays for FcRI-dependent binding, phagocytosis, and antibody-dependent cellular cytotoxicity mediated by monomeric IgG gamma 2a in murine peritoneal macrophages

Mouse peritoneal macrophages possess distinct Fc receptors (FcR) for binding the various murine IgG isotypes. FcRI binds monomeric IgG gamma 2a, but not monomeric IgG gamma 2b or IgG gamma 1 with high affinity at 4 degrees C and is sensitive to trypsin degradation. We have assessed the functional consequences of the cytophilic binding at 4 degrees C of monomeric IgG gamma 2a to FcRI of mouse peritoneal macrophages using newly developed photometric microassays for quantification of binding, phagocytosis, and antibody dependent cellular cytotoxicity (ADCC) of post-opsonized sheep red blood cell (SRBC) targets. Dose-dependent binding specificity of monomeric IgG gamma 2a, but not IgG gamma 2b or IgG gamma 1 to FcRI of oil-elicited mouse peritoneal macrophages at 4 degrees C for 2 h was confirmed to display typical saturation kinetics both by the photometric assay and by a cellular enzyme-linked immunosorbent assay (CELISA). Binding of monomeric IgG gamma 2a to macrophage FcRI promoted highly efficient phagocytosis of opsonized SRBC in that most cells that were bound were also rapidly internalized by the phagocytic process during a 1 h incubation at 37 degrees C. Upregulation of FcRI-dependent binding and phagocytosis occurred during 24-48 h in vitro culture of macrophages as shown both by the photometric assays and CELISA. Trypsin treatment of macrophages abrogated FcRI-dependent binding and phagocytosis by monomeric IgG gamma 2a, but had little effect on FcRII-dependent functions. Cytophilic binding of monomeric IgG gamma 2a to FcRI failed to trigger ADCC activation. Thus functional characterization of macrophage FcRI-dependent effector functions confirmed the fidelity of binding specificity of monomeric IgG gamma 2a to a trypsin degradable receptor which mediates highly efficient phagocytosis but fails to initiate the signal for ADCC activation. It appears that passively bound immune monomeric IgG gamma 2a could provide an efficient mechanism by macrophages in vivo for FcRI-dependent immune clearance of soluble or particulate cellular antigens without elicitation of potentially harmful cytolytic factors associated with ADCC activation.     

Characterization of murine macrophage Fc receptor-dependent phagocytosis and antibody-dependent cellular cytotoxicity during in vitro culture with interferons-gamma, alpha/beta and/or fetal bovine serum

Resident and oil-elicited inflammatory peritoneal macrophages (PM phi) from competent C3HeB/FeJ and genetically deficient C3H/HeJ mice were characterized for their Fc receptor (FcR)-dependent binding, phagocytic and ADCC functions during in vitro differentiation under the influence of mouse recombinant interferon-gamma (rIFN-gamma), interferon-alpha/beta (IFN-alpha/beta) fetal bovine serum (FBS), or in serum-free medium. Freshly cultured resident PM phi from C3HeB/FeJ mice had low levels of FcR-mediated phagocytosis in response to mouse monoclonal IgG gamma 2a, IgG gamma 2b or IgG gamma 1, opsonized sheep erythrocytes as compared to oil-elicited inflammatory PM phi from the same strain. Resident PM phi were uniformly upregulated in their FcR-dependent phagocytosis after 24-48 h in vitro culture with FBS to levels approximating that of freshly cultured inflammatory PM phi which were also further upregulated after 24 h in vitro culture with FBS. Both resident and inflammatory PM phi were upregulated largely by an autostimulatory process in that they increased their FcR-mediated phagocytosis in serum-free RPMI-1640 medium without the addition of rIFN-gamma or IFN-alpha/beta, although FBS further augmented FcR upregulation. A synergistic effect of FBS and rIFN-gamma was required for total reconstitution of FcR-mediated phagocytosis of FcR-incompetent C3H/HeJ inflammatory PM phi in that FBS or rIFN-gamma alone only partially reconstituted FcR function, whereas in combination full reconstitution occurred. Thus, macrophages from competent C3HeB/FeJ mice were upregulated in their FcR-mediated functions largely by an autostimulatory process, presumably dependent on endogenous of IFN-beta, whereas, genetically-deficient C3H/HeJ macrophages required exogenous rIFN-gamma in combination with fetal bovine serum for synergistic reconstitution of FcR functions. The uniform upregulation of FcR-dependent effector functions in vitro appears to provide an efficient system for enhanced immune function during differentiation which may be applicable to in vivo situations.     

Effect of rIFN-gamma on antibody-mediated cytotoxicity via human monocyte IgG Fc receptor II (CD32).

Human monocytes and macrophages express an isoform of IgG Fc receptor II (Fc gamma RII), Fc gamma RIIa. Two allotypic variants of this receptor could be distinguished with respect to their ability to bind murine (m)IgG1 complexes either strongly or weakly, defined as high-responder (HR) and low-responder (LR), respectively. We investigated the effect of recombinant (r)IFN-gamma on the ability of freshly isolated monocytes, and those cultured for 40 h and 9 days, to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Using human erythrocytes (E) sensitized with mIgG1 as target cells, Fc gamma RII was studied selectively. Cells which had been cultured for 40 h exhibit a significantly decreased Fc gamma RII expression, and Fc gamma RII-mediated ADCC activity as compared with freshly isolated monocytes. Co-culture with rIFN-gamma (40 h) reversed this decrease. Short-term rIFN-gamma-cultured cells, and fresh cells express similar numbers of Fc gamma RII, and exhibit comparable Fc gamma RII-mediated ADCC activity. Phagocytic activity was not affected. Prolonged culture of monocytes for 9 days, co-cultured with rIFN-gamma either from day 0 or from day 7, did not affect expression or functional activity of Fc gamma RII. Furthermore, the effects were observed in both HR and LR individuals. Our results show that rIFN-gamma has strong effects on Fc gamma RII-mediated responses specifically during the early stages of monocyte maturation, most likely by affecting receptor expression levels.     

Two different Fc gamma receptor-dependent cytotoxic mechanisms triggered by monoclonal immunoglobulins.

It is known that the receptors for the Fc portion of IgG molecules (Fc gamma R) are widely distributed in cells of the immune system. The expression of Fc gamma R enables monocytes and neutrophils to destroy antibody-coated target cells through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. In addition, the interaction of immune complexes or aggregated IgG with monocytes or neutrophils led to the lysis of nonsensitized target cells in a process known as nonspecific cytotoxicity (NSC). Despite that ADCC and NSC are both triggered through Fc gamma R, the cytolytic mechanism involved in each reaction is different. In this paper we analyze the ability of human monoclonal IgG1, IgG2, IgG3 and IgG4 to induce ADCC and NSC. Our results demonstrate that each IgG subclass is able to induce both, NSC and ADCC, mediated by monocytes or neutrophils, indicating that there is no correlation between IgG subclass specificity and the ability to activate both mechanisms.     

Interferon gamma-treated human macrophages display enhanced cytolysis and generation of reactive oxygen metabolites but reduced ingestion upon Fc receptor triggering

Human monocyte-derived macrophages treated with recombinant IFN-gamma (rIFN-gamma) and control cells were assessed for three distinct effector functions, all mediated by Fc receptors. rIFN-gamma-primed macrophage displayed markedly reduced phagocytosis of IgG antibody-coated erythrocytes. In contrast, antibody-dependent cytotoxicity towards IgG-antibody-coated erythrocytes and IgG-antibody-coated erythrocyte-induced generation of reactive oxygen metabolite production were increased. The decreased phagocytosis was observed microscopically, as well as in a spectrometric and a radiometric phagocytosis assay. Evidence is presented that the observed impairment in phagocytosis is not the result of increased extracellular lysis or intracellular catabolism of IgG-antibody-coated erythrocytes and that it is not observed with particles ingested in an Fc receptor-independent manner. Enhanced production of reactive oxygen metabolites was detected most clearly by measurement of luminol-dependent chemiluminescence. Antibody-dependent cellular cytotoxicity was shown to proceed also under conditions impeding phagocytosis, and rIFN-gamma-treated macrophage exerted enhanced antibody-dependent cellular cytotoxicity under these conditions too. In all three assays, functional alterations were optimally expressed after a treatment with 500 U/ml for 46 hr. Analysis at the single-cell level revealed that the IFN-gamma-induced alterations were expressed by all macrophages and not the property of distinct macrophage subpopulations. This and earlier studies suggest that the modulation of Fc receptor-mediated macrophage effector functions by IFN-gamma is in part a post-receptor-binding event.     

Modulation of human monocyte Fc receptor function by surface-adsorbed IgG.

Fc receptor-mediated phagocytosis was measured with monocytes subjected to various treatments. Monocytes exposed to IgG during their adherence, or after they had adhered to a surface, experienced functional impairment. This was manifested in the requirement of a higher antibody density on target particle for efficient phagocytosis, and in an enhanced susceptibility to inhibition by fluid-phase IgG. The impairment was found to be due to an interaction of IgG adhering to the surface with the Fc receptors. This effect could be induced with monomeric IgG, devoid of IgG aggregates or immune complexes. IgG coatings that resulted in inefficient Clq fixation promoted considerable functional impairment of monocytes within 1 hr. In addition, the prolonged contact of monocytes with polystyrene in the absence of IgG also led to a functional reduction. The study points to a compromised function of phagocytes exposed to artificial surfaces.     

Fcγ receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. FcγR recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for FcγR-mediated phagocytosis, specifically FcγRI(p135) and FcγRIIa isoforms. The low-affinity receptor, FcγRIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, FcγRI has no known signaling motifs, though a functional interaction has recently been demonstrated with the γ chain of the high-affinity receptor for IgE, FcεRI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although FcγRI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either γ chain or FcγRIIa replaces that of FcγRI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either FcγRI or FcγRIIa is replaced with that of CD2, a T cell antigen, indicate that FcγR-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.     

Antibody-dependent cell-mediated cytotoxicity (ADCC) toward human O+ red cells coated with anti-D antibody: comparison between lymphocyte and monocyte ADCC activity

We investigated the antibody dependent cell-mediated cytotoxicity (ADCC) of lymphocytes and monocytes toward human O+ red cells coated with anti-D antibody using a 51Cr release assay. Lysis of sensitized red cells by lymphocytes occurred rapidly, but monocyte-mediated lysis occurred slowly. This difference might be due to postphagocytic 51Cr release by monocytes. ADCC of lymphocytes increased in proportion to the effector cell number, but large amounts of antibodies were required. In contrast, ADCC of monocytes was independent of the effector/target ratio and very small amounts of antibodies could produce red cell lysis. Large amounts of fluid phase IgG were required to inhibit the lymphocyte ADCC, whereas the monocyte ADCC was markedly inhibited by small amounts of IgG. Monocyte-mediated lysis was completely inhibited by the addition of 10% human AB serum, but lymphocyte-mediated lysis was only slightly inhibited. Purified IgG1 and IgG3 were much more inhibitory to the lysis by both effectors than IgG2 and IgG4 (IgG2 greater than IgG4). Erythrophagocytosis also was inhibited by IgG1 and IgG3. These studies demonstrate that lymphocytes as well as monocytes can cause the lysis of antibody sensitized red cells, and IgG1 and IgG3 subclasses are more important than IgG2 and IgG4 in causing lysis of anti-D coated red cells.     

Comparison of receptors required for entry of Leishmania major amastigotes into macrophages.

We investigated the mechanisms of entry of amastigotes of Leishmania major from two different sources into macrophages by comparing their use of the Fc receptor (FcR), complement receptor type 3 (CR3), and mannose-fucose receptor (MFR). Amastigotes were obtained from BALB/c mice and SCID mice. FcR involvement was examined by opsonizing L. major with parasite-specific immunoglobulin G (IgG). Antiparasite IgG did not alter the uptake of amastigotes from BALB/c mice since these amastigotes had antibody bound to their surface: IgG1 was the most predominant antibody, followed by IgG2b, IgM, and IgG2a. However, opsonization with antiparasite IgG enhanced the entry of amastigotes that lacked antibody on their surface, namely, amastigotes obtained from SCID mice or from macrophages infected in vitro. These results indicate that the FcR is important for amastigote entry into macrophages. Down-modulation of FcRs onto immune complexes, however, did not reduce the entry of amastigotes containing surface-bound IgG into macrophages. Monoclonal antibodies against the CR3 inhibited the entry of amastigotes from either BALB/c or SCID mice into J774A.1 macrophage-like cells. Simultaneous blocking of FcR and CR3 further increased the inhibition of phagocytosis. Treatment of macrophages with soluble mannan or down-modulating the MFR onto mannan-coated coverslips had no effect on the entry of amastigotes from BALB/c or SCID mice. Thus, the MFR does not appear to be used by amastigotes of L. major. We show that ingestion of amastigotes appears to occur primarily through the FcR and CR3; however, additional receptors may also participate in the uptake of amastigotes.     

Effect of IgG for intravenous use on Fc receptor-mediated phagocytosis by human monocytes.

Polyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR-mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface-bound IgG displayed impaired phagocytosis of IgG-coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG (2-10 mg/ml). Monocytes pre-exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgG1 and with anti-D-treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR-mediated phagocytosis by monocytes. A flow cytometric analysis using anti-FcRI, FcRII and FcRII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.     

Antibody-dependent cellular cytotoxicity mediated by subpopulations of human T lymphocytes: killing of human erythrocytes and autologous lymphoid cells.

The present study characterized the cytotoxic capabilities of human T lymphocyte subpopulations against human red blood cells (HRBC) and autologous lymphoid cells in an antibody-dependent cellular cytotoxicity (ADCC) assay. T cells bearing Fc receptors for immunoglobulin IgG (TG) were capable of lysis of antibody-coated HRBC and autologous lymphoid cells while T cells with surface Fc receptors for IgM (TM) displayed no ADCC activity TG-cell mediated ADCC could be inhibited by blockage of surface Fc receptors following treatment with aggregated Ig. Null cells and low-affinity E-rosette forming cells were also capable of similar ADCC activity against these targets.     

Complement and cell-mediated lysis of haptenated erythrocytes sensitized with oligomers of rabbit igg antibody

Trinitrophenylated (TNP)‡ chicken erythrocytes were coated with various amounts of radiolabeled, affinity cross-linked oligomers of rabbit anti-TNP IgG. These cells were used as targets for lysis by complement or by several types of effector cells, normal splenocytes, cells of a macrophage line (P388D₁) or cells of two lymphoma lines (PL-1 and ABLS-5).With complement as mediator, lysis increased with the number and size of target-bound molecules. In contrast, antibody-dependent, cell-mediated cytolysis (ADCC) depended only on the number of sub-units bound, and was independent of oligomer size. Moreover, complement-mediated lysis required a threshold level of bound antibody before lysis occurred, whereas at low levels of sensitization ADCC increased linearly with increasing antibody. These results suggest that IgG clusters on the target cell are required for complement-mediated lysis but not for cell-mediated lysis. Furthermore, the data suggest that the crosslinking of neighboring Fc receptors on the surface of effector cells is not required for initiating ADCC.Non-immune IgM and small oligomers of non-immune IgG were used to block lysis mediated by each type of effector. ADCC was readily inhibited by small oligomers but not by IgM, while complement was inhibited by only larger IgG oligomers and by IgM. These results suggest that ADCC is not blocked by IgM, but is much more susceptible to inhibition by small IgG immune complexes than complement-mediated lysis.     

Defective antibody-dependent tumour cell lysis by neutrophils from cancer patients.

Neutrophil antibody-dependent cellular cytotoxicity (ADCC) against Raji target cells was studied in 32 patients with lung or gastro-intestinal carcinoma and 25 healthy controls. Seventeen of the patients (53%) had defective ADCC. Moreover, neutrophils obtained from patients with defective ADCC were found to bind IgG-coated target cells normally. Thus, the defect responsible for the impaired lysis appears to be distal to the Fc receptor (FcR)-mediated target cell binding by neutrophils.     

Regulation of Effector Functions of Human K-Cells and Monocytes by Antigen Density of the Target Cells

Antibody-dependent cytolysis and phagocytosis mediated by human K-cells or monocytes against chicken erythrocytes (ChRBC) were studied. The antigen density of the target cells was varied by coating the cells with different amounts of 3H-labelled dinitrophenyl (DNP) hapten. The degree of antigenicity thus acquired by the target cells was assessed on the basis of their uptake of the isotope. Anti-DNP serum was used to induce lysis or phagocytosis. Below 500 antigenic determinants per ChRBC the target cells were not affected. However, at the density, lysis and/or phagocytosis was seen when the antibody concentration was high (2 X 10(-9) M). With less antibody present (2 X 10(-11) M) only monocyte-mediated phagocytosis was induced. The estimated lowest number of target-cell-bound antibodies required for K-cell-mediated lysis was approximately 50. The corresponding number for monocyte-mediated phagocytosis was approximately 20 IgG per ChRBC. The result suggests that interaction of several Fc receptors on the effector cells with IgG molecules bound to adjacent sites on the target cell membrane is an important factor in the regulation of these antibody-dependent cell-mediated effector functions.     

The Fc Receptors of Normal and Cancer Patients Monocytes

The ability of human monocytes from normal donors and gastric-cancer patients to form rosettes with ?0? Rh⁺(D) human erythrocytes coated with hyperimmune IgG anti-D antibody (EA_hu) and to kill the same target in antibody-dependent cellular cytotoxicity (ADCC) were assessed. Trypsin pretreatment of normal monocytes decreased their ability to form rosettes with EA_hu complexes, but their ADCC activity was unaffected. The Fc receptor (FcR) expression and ADCC activity of monocytes of cancer patients were elevated, and trypsin-treatment led to their further increase. The elevated values were related to the presence of the tumour. These results may suggest that human monocytes possess trypsin-sensitive and trypsin-resistant Fc receptors. The trypsin-resistant FcR seems to be involved in ADCC phenomenon and to be preferentially expressed on monocytes of some cancer patients.     

Macrophage colony-stimulating factor enhances the expression of Fc receptors on murine peritoneal macrophages

Experiments were performed to test the postulate that macrophage colony-stimulating factor (M-CSF) modulates the expression of specific membrane structures on mature murine macrophages. Resident peritoneal macrophages were incubated with M-CSF for 48-72 hr and analysed for Fc receptor and Ia antigen expression. M-CSF treatment of macrophages increased the expression of Fc receptors two- to three-fold over that of unstimulated macrophages. The effect was detected at 1 U/ml of M-CSF, with maximal expression between 100 and 500 U/ml. The specificity of the enhancement was indicated by two sets of experiments. Purified rabbit anti-M-CSF IgG, but not normal rabbit IgG, inhibited the M-CSF-mediated Fc receptor enhancement. Also, a highly purified M-CSF preparation, which was essentially free of endotoxin and interferon, was active in these assays. M-CSF augmented both major types of IgG Fc receptors, FcR I (recognizing IgG 2a) and FcR II (recognizing IgG 2b/IgG1). A second membrane marker, the Ia antigen, was induced by recombinant IFN-gamma (rIFN-gamma) but not by M-CSF. These results indicate that M-CSF is an inducer of two classes of IgG Fc receptors on mature tissue macrophages.     

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.