Phagocytic uptake of synthetic particles in blood leukocytes of fetal and newborn athymic nude mice

Monocytes are the main phagocytic element in the blood of fetal mice; monocytes of athymic nude mouse fetuses (gestation days 17-20) ingest in vitro, in full blood, significantly more synthetic (HEMA) particles than the monocytes of their euthymic littermates. Blood leukocytes of athymic fetuses also bear much higher densities of Fc receptors for IgG2B. The differences in phagocytic uptake in blood leukocytes disappear and the differences in Fc(IgG2B) receptors decrease at postnatal days 2 and 4.     

Cell surface events which may initiate lysosomal enzyme secretion by human monocytes

Freshly isolated and subsequently matured human monocytes secreted lysosomal hexosaminidase in response to exposure to IgG-Sepharose, but not certain derivatized control Sepharoses. The cells bound selectively to the surface of IgG-Sepharose (and not the control Sepharoses) but because of the large size of the particles, could not ingest them. Since the soluble IgG was covalently linked to the Sepharose and free soluble IgG was not an inducer of secretion, the secretion was thus induced directly at the cell surface. Zymosan, a yeast cell wall particle which contains a mannan, was also able to induce secretion in the various monocyte stages under study. It could even bind to the cell surface of fresh monocytes which lacked the receptor for mannose-terminated glycoproteins, and induce secretion in these cells. The mannose receptor appeared as monocytes matured, and the Ir number on the surface was increased by the action of lymphokines. Although zymosan-induced secretion could be inhibited by mannose and certain other sugars, these seemed to have some complex metabolic effects in human monocytes (which previous work with mouse macrophages has not revealed). Thus, it was not possible to demonstrate whether zymosan could initiate secretion directly by interaction at the monocyte surface mannose glycoprotein receptor.     

Fc gamma receptor recognition of IgG ligand by human monocytes and macrophages

We examined the binding characteristics of human monocytes and macrophages with the IgG ligands, human monomeric IgG and a small human IgG aggregate, trimeric IgG. Our purpose was to utilize fresh monocytes, in vitro cultured monocytes, and alveolar macrophages in direct and indirect binding experiments. Freshly isolated monocytes expressed only a single binding site for IgG monomer and IgG trimer. In contrast, in vitro cultured monocytes, gamma-interferon-treated monocytes, and freshly isolated alveolar macrophages expressed a single binding site for IgG monomer and, in addition, a high and low affinity binding site for IgG trimer. The high affinity binding site for IgG trimer (Kd approximately equal to 1 nM) appeared identical to the binding site for IgG monomer. The low affinity binding site for IgG trimer (Kd = 50 to 250 nM) appeared to be due to Fc gamma RII, because antibody to Fc gamma RII inhibited its expression. Since Fc gamma RII, in contrast to Fc gamma RI, does not bind monomeric IgG, the data suggest that this low affinity receptor for trimeric IgG, Fc gamma RII, can bind low molecular weight circulating immune complexes at concentrations 10- to 100-fold lower than Fc gamma RI. Thus, these studies suggest that at 37 degrees C, macrophage Fc gamma RII may play a functional role in the recognition of small molecular weight immune complexes.     

Fc receptor function on sheep alveolar macrophages

We have examined the binding to sheep alveolar macrophages (AM) and peripheral blood polymorphonuclear leukocytes (PMN) of sheep immunoglobulin G subclasses or rabbit IgG immune complexes formed between rabbit anti-DNP IgG and DNP-bovine serum albumin. Binding studies using 125I-rabbit IgG immune complexes demonstrated 6.6 +/- 3.5 X 10(4) receptors per alveolar macrophage; these receptors bound immune complexes with an average association constant of 3.3 X 10(7) M-1. Saturation binding was achieved by 90 minutes at 4 degrees C with 6 X 10(-8) M IgG. Binding of subclasses of sheep IgG was examined by immunofluorescence. Only 10% of alveolar macrophages bound monomeric IgG1 and no binding of sheep IgG2 monomer could be demonstrated. In contrast, most peripheral blood PMN (93.0 +/- 9.5%) bound IgG2, but not IgG1. No binding to adult peripheral blood PMN of rabbit IgG immune complexes could be demonstrated. To study further the development of pulmonary host defense, we examined the expression of receptors for IgG immune complexes (Fc gamma R) on alveolar macrophages obtained from animals aged 8 through 180 days. At 8 and 21 days of age, the number of Fc gamma R varied considerably (75,000-192,000 sites per cell) and equalled or even exceeded that of adult sheep. Fc gamma R number declined by 42 and 90 days of age, where a nadir was reached (37,000 +/- 6,000 and 25,000 +/- 6,000 sites, respectively). By 180 days of age, the number of receptors had approached those of normal adult sheep (70,000 +/- 20,000 sites per cell). These studies parallel previous observations that revealed age-related differences in the phagocytic capacity of ovine alveolar macrophages.     

The streptococcal protease IdeS modulates bacterial IgGFc binding and generates 1/2Fc fragments with the ability to prime polymorphonuclear leucocytes

The important human bacterial pathogen Streptococcus pyogenes has evolved a variety of mechanisms to evade the actions of the human immune system. M protein and M-like proteins are major virulence factors that bind with high affinity to the Fc-part of IgG. However, the contribution of non-immune binding of IgG to bacterial virulence is not fully established. Importantly, the capacity of S. pyogenes to bind IgG is limited and due to the presence of large amounts of IgG present in vivo, the majority of IgGFc binding sites at the streptococcal surface are likely to be occupied by non-specific IgG. S. pyogenes also secretes a highly effective IgG-endopeptidase, IdeS that inhibits phagocytic killing by cleavage of specific IgG creating F(ab')2 and 1/2Fc fragments. In the present work, IgG and 1/2Fc binding to the streptococcal surface was studied and correlated to IdeS activity. Binding of IgG to the streptococcal surface is shown to be equilibrium and thus not designed to mediate a lasting protection against specific antibodies. However, non-immune binding of IgG to the bacterial surface is followed by the proteolytic cleavage of the antibody by the IgG-endopeptidase IdeS. IdeS generated 1/2Fc fragments do not compete efficiently with intact IgG in binding to the bacterial surface and rapid dissociation of 1/2Fc allows binding of new IgG. Thus, a correlated binding and proteolytic cleavage of IgG also increases the probability that the bacteria can resist specific IgG, despite the presence of a large excess of non-specific IgG in the circulation. As a consequence of IdeS activity, circulating 1/2Fc fragments are generated. These 1/2Fc fragments were shown to be biological active by acting as priming agents for polymorphonuclear leucocytes, suggesting a new mechanism of immune evasion employed by S. pyogenes.     

The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte Fc receptors

Guinea-pig IgG2 and IgT1 bind to contiguous Fc receptors on homologous peritoneal macrophages. Equilibrium association constants determined for the binding of human IgG subclasses to homologous peripheral blood monocytes show that the order of binding is IgG1 greater than IgG3 greater than IgG4 greater than IgG2. Direct binding and rosette assay techniques independently established that both guinea-pig IgG2 and human IgG bind to homologous macrophage-monocyte Fc receptors through a site present in whole Fc (CH2. CH3)2, but absent in pFc' subfragments (CH3)2.     

Phagocytic cells internalize ZnO particles by FcγII/III-receptor pathway

The present study investigates the process of internalization for bulk ZnO particles in macrophages, and further elucidates the underlying mechanism. Since macrophages are active phagocytes and phagocytosis is a size dependent phenomenon, therefore we hypothesized that bulk ZnO may internalize into macrophages by phagocytic pathways. Interestingly, the phagocytic activity got enhanced in bulk ZnO treated macrophages. Moreover, the bulk ZnO treated macrophages internalized via FcγR-II/III, complement and scavenger–receptor pathways. To confirm the specificity of phagocytic pathway, the uptake was also analyzed in splenocytes where phagocytic (monocytes) and non-phagocytic cells (lymphocytes) are present. It was observed that no significant uptake of bulk ZnO in case of lymphocytes whereas significant uptake in monocytes. Henceforth, our quest for uptake mechanisms also revealed that severe plasma membrane extensions (pseudopodia), FcγR clustering over the surface of macrophages and activation of FcγR signaling were the key players for bulk ZnO uptake; whereas clathrin or caveolae mediated endocytic pathways contributed less. Uptake of these particles was further strengthened by the ZnO-induced activation of the Src-kinase p-Lyn, phospho-tyrosine kinases Syk (spleen tyrosine kinase), p-PLC-γ and PI3K (phosphatidylinositol 3-kinase). Our findings illustrate that the phagocytic nature of macrophages could have led to higher uptake of bulk ZnO.     

The functional activity of human monocytes passively sensitized with monoclonal anti-D suggests a novel role for Fc gamma RI in the immune destruction of blood cells.

The role of Fc gamma RI in the immune destruction of blood cells is uncertain as serum IgG levels are sufficient to competitively inhibit interactions between this high-affinity receptor and sensitized red cells. In the current study, it is proposed that, rather than functioning as a receptor for opsonized red cells, Fc gamma RI might, under appropriate conditions, mediate the passive sensitization (or 'arming') of human macrophages with IgG antibodies resulting in the in vivo destruction of unsensitized cells expressing the corresponding antigen. To examine this hypothesis, Fc gamma RI-bearing human monocytes and U937 cells were first passively sensitized by incubation in vitro with human monoclonal anti-D, and then incubated with D-positive red cells. The uptake of monoclonal anti-D by U937 cells was rapid and, in the presence of 2.5 micrograms/ml IgG1 or IgG3 anti-D, was almost complete after 5 min at 37 degrees. Subsequent incubation of passively sensitized U937 cells in an IgG-free medium for 1 hr at 37 degrees resulted in the loss from the cell surface of approximately 50% cell-bound IgG; the remaining cell-bound IgG was lost more slowly despite repeated washing. In functional assays, passively sensitized monocytes (M-IgG) mediated adherent, phagocytic and chemiluminescent (CL) responses to D-positive red cells. After incubation of M-IgG in 50% v/v fresh normal human serum (FNHS) for 2 hr, sufficient anti-D remained bound to monocytes to promote the adherence of red cells. The adherence and phagocytosis of red cells by M-IgG was enhanced by the simultaneous addition of 50% FNHS, probably owing to the binding of low levels of C3bi to red cells. In contrast, phagocytic and CL responses of unsensitized monocytes to anti-D-sensitized red cells (E-IgG) were abrogated in the presence of 0.25% v/v FNHS, presumably owing to blocking of Fc gamma RI by IgG. It is considered that in vivo, Fc gamma RI may mediate the passive sensitization of macrophages in close proximity with antibody-secreting cells in the reticular network of the splenic cords. Once 'armed' in this way, macrophages may destroy cells expressing the appropriate antigen.     

Phagocytosis of yeast: a method for concurrent quantification of binding and internalization using differential interference contrast microscopy

In studies of phagocytosis there is a need to distinguish targets that are internalized by the cell from those that are bound to the cell surface. The present work describes a simple method by which internalized and surface-bound yeast particles can be identified by differential interference contrast microscopy, using trypan blue to stain surface-bound yeast particles. The method has the advantage that both internalized and surface-bound particles can be visualized without the need to switch the illumination source and/or filter sets, thus facilitating concurrent quantitation of binding and internalization. The method was evaluated with the phagocytosis-modulating agents horseradish peroxidase (HRP) and cytochalasin D, using adherent resident macrophages as phagocytic cells. When macrophages are challenged with a particular type of target, they usually bind many more targets than they ingest. It was shown that yeast particles were arrested in the initial binding phase of phagocytosis depending on the region of macrophage plasma membrane where binding sites were formed. Failure of surface-bound yeast particles to trigger internalization was not due to modifications of the yeast particle surface. Nor was it due to binding to non-phagocytic receptors, or low-affinity receptor-ligand interactions. The glycoprotein HRP inhibited only the binding stage of phagocytosis, whereas cytochalasin D, a drug that affects actin polymerization, inhibited both binding and internalization. However, when the yeast particles were pre-incubated in fresh mouse serum, cytochalasin D inhibited only the internalization step. The assay described here may be useful in studies concerned with the function and expression of phagocytosis-mediating surface lectins.     

The spontaneous ability of normal human IgG to inhibit the Fc receptors of normal human monocytes is related to their binding capacity to lectins

The lectin-binding capacity of 96 normal human IgG, assessed by solid-phase radioimmunoassay, strikingly varied according to the lectin considered. Indeed, half of the IgG samples exhibited peanut agglutinin (PNA)- and pokeweed mitogen-specific binding capacities superior or equal to 4%, whereas less than 15% of IgG similarly bound to concanavalin A (Con A) and to phytohemagglutinin. The ability of those IgG to inhibit the Fc receptor (Fc-R) function of human monocytes, measured by a classical rosette assay, was inversely correlated to their binding ratios to PNA and Con A only. By affinity chromatography, three groups of IgG were separated: the IgG purified on agarose-PNA columns slightly reduced the Fc-R function (40-45% inhibition); the IgG purified on Sepharose-Con A columns exhibited the highest inhibitory properties (80-85% inhibition); the IgG that did not bind to PNA and Con A columns possessed intermediate inhibitory properties (65-70% inhibition). The different effect of IgG on Fc receptors was conserved when monocytes were first treated by trypsin and was unrelated to their specific binding to human monocytes, to their subclasses, and to their C1q- or protein A-binding capacities. Incubation of monocytes with D-galactose (10 mM) significantly improved their capacity to form IgG rosettes, whereas their incubation with D-mannose (10 mM) significantly reduced the Fc-R function. Scatchard plots of 125I-IgG1 myeloma protein binding to monocytes were linear under basal conditions, as well as after a prior incubation of the cells with D-galactose or D-mannose. Monocytes bound about 16,000 molecules of IgG1 per cell in each instance. In contrast, the mean association constant (Ka) for IgG1 binding was 2.59 +/- 0.50 X 10(8) M-1 under basal conditions, 4.4 +/- 0.75 X 10(8) M-1 after D-galactose incubation, and 1.35 +/- 0.50 X 10(8) M-1 after D-mannose incubation. These data suggest that the level of human monocyte Fc-R function blockade induced by human IgG depends mainly on the presence of "accessible" galactosyl or mannosyl residues in the Fc domain and that the modulation of the Fc-R function induced by these carbohydrates is due to a change in the affinity rather than in the number of single class of high-affinity binding sites.     

Expression of Fc Gamma Binding Sites on Human Extraplacental Membranes*

ABSTRACT: Binding sites for the Fc portion of immunoglobulin G (IgG) molecules (FcγBS) were identified on amnionic epithelial cells and chorionic trophoblast cells in human extraplacental membranes using direct immunofluorescence with labeled human IgG. The binding sites are similar to FcγBS on placental trophoblast in their specificity and high degree of relative binding affinity for IgG monomers. Neither immunoglobulin A (IgA) nor IgM bound to a significant degree to the membranes. Binding was completely inhibited with unlabeled IgG but not F(ab')2 fragments of IgG. Studies on isolated amnion cells demonstrated that the binding sites are expressed on the cell membrane. At the level of sensitivity of the immunohistologic methods used, aggregated IgG or antigen-antibody complexes failed to bind amnionic epithelial cells or chorionic trophoblasts. This is in contrast to FcγBS on macrophages which, being present in the same tissue, failed to exhibit significant binding of deaggregated IgG but bound complexes avidly.     

The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.

Two different methods, a rosette assay and a direct binding assay, have been employed in an examination of the binding of human IgG1 to mouse macrophages. In both cases, inhibition of IgG binding was demonstrated by Fc (CH2 + CH3 domains) and pFc' (CH3 domains) fragments of human IgG. In a homologous system, the binding of 125I-human IgG to human peripheral-blood monocytes was inhibited by the Fc fragment whereas the pFc' fragment was inactive. Scatchard plot analysis of the binding data from both the heterologous and homologous systems allowed association constants and numbers of receptors per cell to be calculated. A more thorough examination of the possible location of IgG Fc-receptor binding sites was made using less orthodox proteolytic cleavage fragments of IgG. The site on human IgG1 responsible for binding to mouse macrophage Fc receptors was confirmed to be within the CH3 domains. Human IgG1 binding to homologous monocytes was shown, using a dimeric C gamma 2 domain fragment, to be via the CH2 domains, and was dependent on the integrity of the covalent interaction between the C gamma 2 domains at the hinge region.     

Interferon-gamma restores T lymphocyte proliferation of nonresponders to IgG1 anti-CD3 via the induction of Fc gamma 1 receptors on monocytes

Human peripheral blood T lymphocytes are stimulated to grow and divide by some mouse anti-CD3 monoclonal antibodies. This polyclonal mitogenesis is dependent on both their immunoglobulin subclass and the presence of monocytes. The unresponsiveness of T lymphocytes from certain individuals to mouse IgG1 (or IgG2a) antibodies is due to a failure of their monocytes to bind these IgG isotypes. In this study, we have selected such nonresponder subjects to IgG1 anti-CD3 (UCHT 1) in order to study their monocytes. Two assays were used: IgG1 and IgG2 EA rosettes to evaluate their Fc receptor-binding capacity, and IgG-mediated monocyte chemiluminescence to test their receptor-related activation since mouse anti-T cell antibodies binding to lymphocytes trigger monocyte chemiluminescence via their Fc receptor. We have observed that in all nonresponder subjects the absence of IgG1 anti-CD3 monocyte chemiluminescence strictly correlates with the absence of IgG1 EA rosettes. Thus, the failure to respond to UCHT 1, in all nonresponders tested to date, is due to the absence of Fc gamma 1 receptors on their monocytes. Treatment of nonresponder monocytes by recombinant interferon-gamma was shown to restore T cell proliferation and monocyte chemiluminescence in nonresponders. This effect of interferon-gamma correlates with the appearance of Fc gamma 1 receptors on monocytes from these individuals. This work strongly suggests that nonresponder monocytes possess functional genes for Fc gamma 1 receptors which are not expressed normally at a detectable level but can be induced by interferon-gamma.     

Enhancement of hamster alveolar macrophage phagocytic activity by lymphokines

Modulation of phagocytic activity of resident hamster pulmonary alveolar macrophages was accomplished by incubation of the cells in lymphokines prepared by stimulation of hamster splenocytes with concanavalin A or alloantigens in mixed lymphocyte cultures. Alveolar macrophages preincubated in either of these lymphokine preparations possessed significantly greater ability to ingest IgG or IgM plus complement-coated sheep erythrocytes, via their Fc or complement receptors, respectively, than macrophages exposed to control preparations. Ingestion of yeast particles also was enhanced with macrophages incubated in supernatants from cultures of stimulated splenocytes. Supernatant fluids from either mitogen- or alloantigen-stimulated splenocytes possessed migration inhibitory activity with characteristics similar to MIF from other animals; the phagocytosis-enhancing activity shared some of these characteristics.     

The human FcR. III. Effects of pronase on soluble immune complex binding by polymorphonuclear leucocytes, monocytes and pulmonary macrophages.

The effect of Pronase incubation on the Fc receptors of human polymorphonuclear leucocytes (PMN), monocytes and pulmonary alveolar macrophages was evaluated by Scatchard analysis of the binding of soluble immune complexes at equilibrium. All three cell types, when preincubated with Pronase, demonstrated a significant increase in Fc receptor affinity. Maximum binding (which measures the number of Fc receptors) on polymorphonuclear leucocytes was reduced 45%-50% but was unchanged on monocytes and pulmonary macrophages. The changes in Fc receptor affinity and maximum binding of the PMN were reversible in short-term culture, an effect which was prevented by cycloheximide. These studies indicate that the affinity of the Fc receptor of human phagocytic cells may change significantly independent of changes in receptor number and that this effect can be caused by extracellular proteases. In addition, the human polymorphonuclear leucocyte demonstrates a subpopulation of Fc receptors which is decreased by Pronase and which recovers, in vitro, by a process requiring protein synthesis.     

Distinctive role of IgG1 and IgG3 isotypes in Fc gamma R-mediated functions

Polyclonal and monoclonal anti-Rh (D) antibodies of IgG1 and IgG3 subclass were evaluated for their capacity to sensitize erythrocytes and (i) to trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC); (ii) to mediate binding to monocyte and lymphocyte Fc gamma R; (iii) to stimulate phagocytosis by monocytes. All antibodies were equally effective in mediating monocyte or activated U937 cell ADCC but IgG1 was more active than IgG3 in K-cell mediated ADCC. IgG3-sensitized erythrocytes inhibited IgG1-induced lysis, suggesting that each subclass engages the same Fc gamma R receptor but that lysis requires a further 'signal' that the IgG3 molecule can not deliver. Two monoclonal IgG3 anti-D antibodies were shown to have higher binding (two times) and phagocytic (three times) indices than IgG1 antibody for monocytes; similar differences were observed for polyclonal IgG1 and IgG3 antibodies. The same pattern was observed in an EA rosette assay when a total lymphocyte population was used; however, this difference was not seen with a B-cell depleted (T+ null cell) lymphocyte population.     

Recombinant Fc of human IgG1 prepared in an Escherichia coli system escapes recognition by macrophages

The Fc portion of IgG is recognized by macrophages via Fc receptors on their cell surface. The recognition of human IgG1-Fc by macrophages was compared for the native and recombinant proteins by a novel assay method using competitive inhibition of the aggregated IgG-mediated chemiluminescence reaction of human macrophage cell line THP-1. Recombinant Fc proteins, prepared in an Escherichia coli system, which lack oligosaccharides and retain normal staphylococcal protein A reactivity and, to a lesser extent, C1q binding capacity, showed inhibitory ability greater than 500 times lower than that of native Fc fragments obtained by proteolytic digestion of myeloma proteins. This suggests that the oligosaccharide chains on IgG1-Fc molecules may be critical for recognition by Fc receptors on macrophages.     

Comparison of the Binding of Radiolabelled Human IgG and Fc Fragments to Murine Spleen Cells

The binding properties of an IgG1 human myeloma protein, normal IgG, and the Fc and Fab fragments of each were compared in cultures of murine spleen cells. Both 125I-labelled IgG and Fc fragments bound to splenic lymphocytes, whereas Fab fragments did not bind significantly at the highest concentrations tested. On a molar basis, more Fc bound than intact IgG. According to Scatchard plot analysis, the affinity constand of IgG1 was 1.5 x 10(6) +/- 1 x 10(5) L/M and that of the Fc fragments was 7.8 x 10(5) +/- 2.6 x 10(5) L/M. Approximately 25,000 binding sites/cell were calculated for IgG1 and 102,000 for Fc. Deaggregation of the Fc preparation did not change these values, suggesting that the difference in binding of IgG and Fc did not result from Fc aggregation. Unlabelled IgG inhibited about 25% of the labelled Fc binding, whereas unlabelled Fc inhibited approximately 80% of the labelled Fc binding. IgG antigen-antibody complexes, however, inhibited 75% of the Fc binding. In the reciprocal experiment both intact IgG and Fc inhibited the binding of labelled IgG by 100%. The major cell population that bound IgG and Fc fragments in the spleen cell preparation were the B lymphocytes. Removal of macrophages did not significantly affect the binding of labelled Fc fragments. In addition, T-cell-enriched populations bound an insignificant quantity of Fc fragments.     

Activation of complement by soluble IgG aggregates and their subsequent interaction with macrophages

The mononuclear phagocyte system (MPS) plays an important role in the removal of both particulate and soluble immunogenic material from the circulation. E.IgG adhere to mononuclear phagocytes if the Fc portion of the IgG can interact with the phagocytes' Fc receptors. Simultaneous sensitization with IgG and C3b enhances the effectiveness of binding and ingestion. If soluble material cannot adhere to the surface of macrophages, it will be endocytosed in vitro via fluid-phase pinocytosis at the concentration that is present in the medium. If the material adheres to the cell's surface via its chemical properties or via specific receptors, it will be selectively concentrated at the cell's surface and endocytosed by an adsorptive pinocytosis. Ingestion of IC via Fc gamma R and C3b depends on the ability of the antibodies to interact with Fc gamma R and their capacity to activate the complement system. IC-bound C3b enhances the adsorptive pinocytosis of IC. Soluble AIgG are also pinocytosed more efficiently when C3b is bound to AIgG. The degree of endocytosis varies with the level of C3b sensitization. The highly effective C3b-mediated pinocytosis can be abolished by treating the macrophages with trypsin to inactivate C3bR. This observation illustrates that C3-mediated pinocytosis can replace Fc-mediated pinocytosis in unstimulated macrophages. Soluble IC and AIgG are removed from the circulation mainly by hepatic Kupffer cells. It seems that the size, the Ag/Ab ratio, the capacity of the IC to bind C1, activate C1, and allow deposition of C3b together with the degree of phagocyte activation determine the degree of binding and subsequent degradation of soluble IC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible errors in the calculation of cell-binding parameters exemplified by the analysis of the IgG-Fc receptor system

The binding parameters of monomeric and polymeric (immune complex with a molecular weight of 500,000 daltons) rabbit IgG to homologous Fc receptor-bearing alveolar macrophages were estimated, using corrected values for IgG and cell concentrations. Considering the maximum percentage of monomeric IgG binding to cells (2.7%) and the maximum percentage of cells binding monomeric IgG (32%) instead of the IgG and cell concentrations in the initial reaction mixture, a 36-fold increase of the equilibrium constant (K) (from 0.6 x 10(6) L/M to 21.3 x 10(6) L/M) and a 3-fold increase of the maximum number of IgG molecules able to bind to a single cell (n) (from 7.8 x 10(5) to 23.7 x 10(5] were registered. Since more than 60% of the polymeric IgG is bound to 46% of the macrophage population, the values of K (from 10.8 x 10(6) L/M to 15.6 x 10(6) L/M) and n (from 4.3 x 10(5) to 9.4 x 10(5] are only doubled by using the corrected values for IgG and cell concentrations. It results that the cytophilic fraction of the monomeric IgG, representing only 2.7% of total IgG, has a slightly higher affinity for the Fc receptors than the polymeric IgG. By considering the actual number of macrophages which bind IgG it is evident that the number of Fc receptors per cell is higher than that determined by the usual procedure which does not take into account cellular heterogeneity.