Cytophilic binding of IgE to the macrophage. III. Involvement of cyclic GMP and calcium in macrophage activation by dimeric or aggregated rat myeloma IgE

Discharge of lysosomal enzymes, measured by release of β-glucuronidase, was studied in uninduced rat macrophages stimulated in vitro with rat monoclonal IgE (IR 162) in different states of aggregation. Monomeric IgE showed negligible activity, while dimeric and aggregated IgE were shown to induce a rapid and selective release of β-glucuronidase as well as new synthesis of the enzyme, without change in the cytoplasmic marker, leucine aminopeptidase. Lysosomal enzyme release is related to the dose of dimeric IgE, becoming maximal above 2.5 μg/ml. β-Glucuronidase release from macrophages by dimers is competitively inhibited by monomeric IgE but only at high ratios, approximately 100-fold greater than those needed to block mast cell release of the same enzyme. The difference in inhibitability is consistent with the difference in binding affinity of macrophages and mast cells for monomeric IgE. This observation rules out the participation of the few remaining mast cells contained in the macrophage monolayer in β-glucuronidase release. Dimeric or aggregated IgE produced a rise in cyclic GMP coincident with the peak fixation of IgE by macrophages. Elevation of cyclic GMP by pharmacological means also stimulated β-glucuronidase release and new synthesis, as well as enhancing the effect on these of aggregated IgE. Enzyme release by IgE did not occur in the absence of extracellular calcium. We conclude that IgE, which has been cross-linked to form dimers before binding to specific macrophage receptors, triggers the cell and that cyclic GMP (and perhaps calcium) modulates the early step of macrophage activation.     

Characteristics of macrophage cytotoxicity induced by IgE immune complexes

In earlier studies, the specific adherence of normal rat macrophages to Schistosoma mansoni schistosomula, followed by macrophage cytotoxicity against the larvae, was shown to be induced by incubation of the macrophages with serum from infected rats containing complexes of IgE antibody and circulating schistosome antigens. By the use of a chromium-51 release assay, it is pointed out that this cytotoxic process is a two-step phenomenon. The first step, i.e., activation of normal unstimulated macrophages induced by incubation of the cell with IgE complexes in immune rat serum, is a nonspecific mechanism which may also be elicited by various other macrophage activators. The second step, i.e., immune adherence and cytotoxicity of activated macrophages against S. mansoni schistosomula, is a specific process which imperatively needs the presence of S. mansoni IgE immune complexes. Aggregated myeloma IgE does not activate adherent peritoneal cells into cytotoxic effector cells unless the further participation of these specific IgE immune complexes is provided. The necessary preincubation of macrophages with immune rat serum before adding schistosomula accounts for the inefficiency of the incubation of the target itself with serum to elicit macrophage cytotoxicity. Serum dilution also appears as a critical factor since immune rat serum is inefficient when diluted more than $\text{1}\text{25}$. Aggregated rat IgG neither induces macrophage activation nor inhibits the activation by IgE immune complexes. Though the binding of IgE to the macrophage appears to be isotype specific, homologous immune complexes of IgE antibody and schistosome antigens are required to induce killing of S. mansoni larvae. The possible mechanism of this new model of macrophage activation and cytotoxicity is discussed.     

Cytophilic binding of IgE to the macrophage. I. Binding characteristics of IgE on the surface of macrophages in the rat.

Involvement of IgE antibody in macrophage cytotoxicity against Schistosoma mansoni schistosomula suggests a cytophilic interaction of this class of antibody with the membrane of macrophages. Peroxidase-labeled monoclonal IgE protein was used to investigate IgE-macrophage interaction in the rat. Benzidine-aggregated rat IgE bound to the surface of peritoneal macrophages under experimental conditions, preventing endocytosis of the labeled aggregates. Binding was inhibited by preincubation with unlabeled IgE (aggregated). When unaggregated IgE was used, labeling of the macrophage surface, even when endocytosis was inhibited, was also observed at 37 °C but not at 4 °C. This result indicated different binding characteristics than reported for cytophilic IgG. Radiolabeled monoclonal IgE (deaggregated by ultracentrifugation after labeling) bound to peritoneal rat macrophages at 37 °C with a maximum between 10 and 20 min and a progressive shedding thereafter, whereas no change in bound radioactivity was observed at 4 °C or after preincubation with unlabeled IgE. Radiolabeled bovine serum albumin as a control did not interact significantly with the macrophages at both temperatures in these experimental conditions. The use of ?-mono-specific rabbit anti-rat IgE allowed the identification of IgE on the surface of peritoneal macrophages from rats infected with S. mansoni.     

Interaction between macrophages and Schistosoma mansoni schistosomula: Role of IgG peptides and aggregates on the modulation of β-glucuronidase release and the cytotoxicity against schistosomula

Discharge of lysosomal enzymes, measured by release of β-glucuronidase and cytotoxicity against Schistosoma mansoni schistosomula, was studied when rat macrophages were incubated in the presence of either IgG peptides, resulting from the cleavage of nonimmune IgG by parasitic proteases, or nonimmune aggregated IgG. With peptides, the macrophage activity showed a dramatic decrease while they were stimulated by IgG aggregates. In contrast, the synthesis of lymphocyte activating factor by macrophages was unaffected. The hydrolysis of IgG is carried out by two distinct enzymatic molecules released into the medium by the larvae. The mechanism by which nonimmune IgG peptides or aggregates inhibit or stimulate macrophage activity, regulated by both parameters indicated above, is discussed and is suggested as a general regulation mechanism for the macrophage activity required for parasite survival in the host.     

In vitro binding of an IgE protein to human platelets

Bronchoconstriction in extrinsic asthma is initiated by mediators released from IgE-sensitized leukocytes after contact with polyvalent antigen. Because platelets also contain soluble mediators that can cause bronchoconstriction, platelet activation and release of the contents of platelet granules may play a role in IgE-mediated responses under some circumstances. We therefore sought to determine if platelets are capable of binding IgE and if cross-linking this cell-bound IgE initiates secretion of platelet granule contents. Platelets from 10 normal donors were studied by using automated fluorescence analysis and fluorescence microscopy. We detected binding of a purified myeloma IgE protein to 24.1 +/- 9.6% (mean +/- 2 SD) of the gel-filtered platelets from these normal individuals. Although we could detect the binding of IgE and anti-IgE to a minority of cells, every normal individual had a population of platelets that bound IgE. The amount of IgE that bound to normal platelets appeared to be distributed heterogeneously among the IgE-positive platelet population. Platelets from two individuals with type II Glanzmann's thrombasthenia bound normal amounts of heat-aggregated IgG, but less than 3% of the platelets bound detectable IgE. Moreover, a combination of monoclonal antibodies to glycoproteins IIb and IIIa inhibited the binding of the IgE protein to normal platelets but did not affect the binding of aggregated IgG. Thus, the binding of IgE to human platelets appeared to require the presence of the glycoprotein IIb-IIIa complex. Binding of monomeric IgE to platelets, by itself, did not initiate either platelet aggregation or release of 14C-serotonin. However, both aggregation and secretion of serotonin followed the addition of anti-IgE to IgE-sensitized platelets. These studies indicate that human platelets can bind an IgE myeloma protein in vitro and that cross-linking of surface-bound IgE with anti-IgE initiates aggregation and secretion. If platelets have a similar capacity to bind normal IgE in vivo, it is possible that platelets may participate directly in several atopic or inflammatory disorders in man mediated by this class of antibody.     

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.     

PMA activation of macrophages alters macrophage metabolism of aggregated LDL

Aggregation of LDL may contribute to its retention in atherosclerotic lesions. Previously, we showed that aggregated LDL induces and enters surface-connected compartments (SCCs) in human monocyte-derived macrophages by a process we have named patocytosis. Aggregated LDL was disaggregated and released from SCCs of macrophages when exposed to human lipoprotein-deficient serum. The serum factor that mediated aggregated LDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. We now show that activation of macrophages with PMA inhibits plasmin-mediated release of aggregated LDL from macrophages. With macrophage activation, plasminogen released about 60% less cholesterol and 63% less TCA-insoluble (125)I-aggregated LDL than when macrophages were not activated. Electron microscopy showed that PMA did not cause SCCs to close, which could have trapped aggregated LDL within the SCCs and limited protease access to aggregated LDL. Rather, PMA decreased macrophage generation of plasmin by 61%, and stimulated lysosomal degradation of aggregated LDL by more than 2-fold. Degradation was mediated by protein kinase C, shown by the finding that degradation was inhibited by the protein kinase C inhibitor G?6976. PMA-stimulated degradation of aggregated LDL was associated with a 3-fold increase in cholesterol esterification, consistent with hydrolysis and re-esterification of aggregated LDL-derived cholesteryl ester. In conclusion, macrophage activation with PMA causes more of the aggregated LDL that enters macrophage SCCs to be metabolized by lysosomes. This results in more cholesterol to be stored in macrophages and less aggregated LDL to be available for plasmin-mediated release from macrophage SCCs.     

Role of specific IgE antibodies in peroxidase (EPO) release from human eosinophils

After the demonstration of cytophilic IgE immunoglobulins (Ig) on human blood and lung eosinophils, their role in cell activation was studied by eosinophil peroxidase (EPO) assay. Hypodense human eosinophils from filariasis-infected patients were activated by anti-human Ig or various antigens. A selective release of EPO occurred after incubation with anti-human IgE, but not with anti-human IgG. The activation by antigens showed a strict antibody specificity of cytophilic IgE antibodies. The direct involvement of IgE antibodies in activation by the specific antigen was evidenced by inhibition experiments with aggregated human IgE myeloma protein. Circulating IgE antibodies exhibiting the same specificity and able to induce EPO release were detected in the sera from filariasis patients by a passive sensitization assay. Only the hypodense eosinophils were able to release EPO after IgE-dependent activation both in the direct assay and in the passive sensitization test, confirming the functional heterogeneity of human eosinophils. These results suggest that the interaction between IgE antibodies and human eosinophils can play a role both in protective immunity and pathology by releasing active pharmacologic mediators.     

Triggering of cultured neoplastic mast cells by antibodies to the receptor for IgE.

Cell surface receptors for IgE were isolated from detergent lysates of iodinated, IgE-saturated, rat basophilic leukemia cells by precipitation with anti-IgE antibodies followed by chromatography at acid pH. The isolated material showed a single 125I-band (m.w. approximately 58,000) on gel electrophoresis in sodium dodecyl sulfate and was used to immunize a rabbit. The resulting anti-serum was reacted with lysates of surface iodinated mouse or rat tumor mast cells. Analysis of the precipitates on (10%) gel electrophoresis revealed one major peak comprising greater than 80% of the detectable counts and having an estimated m.w. of approximately 58,000. The antiserum reacted with detergent-solubilized and cell-bound receptors in the presence or absence of excess IgE; it also inhibited the binding of 125I-IgE. Cultured mouse mastocytoma cells never exposed to IgE released 3H-serotonin when incubated with F(ab')2, but not Fab' fragments of the antiserum, which had been rigorously freed of IgE and anti-IgE. The release was inhibited in the presence of excess IgE, was Ca++ dependent, and equaled 80% of the maximum obtained with IgE and anti-IgE. We conclude that aggregation of the receptors for IgE provides the critical signals for cell activation.     

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.     

Extracellular release of rat eosinophil peroxidase (EPO) I. Role of anaphylactic immunoglobulins

The release of intracellular peroxidase (EPO) was investigated in order to evaluate rat eosinophil activation by various immunoglobulin (Ig) isotypes. After successive incubations with purified rat IgG1, IgG2a, IgG2b, IgG2c, IgE, or IgM and their respective anti-Ig antisera, eosinophils released significant amounts of EPO (up to 26% of the intracellular content) only in the case of Ig with anaphylactic activities (IgG2a and IgE). Other classes and subclasses were unable to induce EPO exocytosis. Selective depletion and reconstitution experiments suggested that mast cells were not required in this process. Similar levels of EPO could be released after interaction of eosinophils with antigen-antibody complexes (IgG2a monoclonal antibody and Schistosoma mansoni antigen) immobilized on nonphagocytosable surfaces. These results indicate that EPO exocytosis can be obtained after cell activation with specific antibodies, and that this mechanism is independent of phagocytosis. A kinetic study of eosinophils from S. mansoni-infected rats revealed that IgG2a and IgE cytophilic antibodies induced EPO release after incubation with either specific antisera or specific antigen, which suggests the in vivo relevance of such findings. The present work underlines the parallelism of interaction of anaphylactic-type Ig with eosinophils and with mast cells. Moreover, EPO release seems to represent an interesting marker of eosinophil activation, because close relationships were established between the present findings and previous work on the effector function of rat eosinophils.     

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.     

Anti-IgE autoantibody in patients with atopic dermatitis

The anti-IgE autoantibody in the IgG class was detected in 39/45 (86.7%) patients with atopic dermatitis by using a newly established solid-phase enzyme immunoassay. The epsilon-chain specificity of the anti-IgE autoantibody was confirmed by competitive inhibition by using human IgG, IgA, IgM, IgD, IgE, and heat-denatured IgE protein. Significant correlations were observed between the levels of the anti-IgE autoantibody and the serum IgE. Gel filtration studies indicated that the anti-IgE autoantibody in sera from atopic dermatitis was mainly present in the form of an immune complex with self IgE. The role of the IgE-anti-IgE immune complex and the role of the anti-IgE autoantibody in the modulation of the IgE-mediated immune system in atopic dermatitis are discussed.     

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.     

Human lung macrophage-derived histamine-releasing activity is due to IgE-dependent factors

Human lung macrophages obtained from surgical specimens spontaneously secreted a factor(s) (which we term macrophage factor) during 24-hr culture that induced calcium-dependent histamine release from human basophils and lung mast cells. Macrophage factor induced noncytotoxic histamine release from purified (85%) basophils. The kinetics of release were relatively slow and similar to that of anti-IgE. We performed a series of experiments to test the IgE dependence of macrophage factor-induced release. Preincubation of basophils with anti-IgE in calcium-free medium resulted in complete desensitization to macrophage factor-induced histamine release (i.e., when calcium and macrophage factor were added to the basophils, no histamine release occurred), and preincubation with macrophage factor in calcium-free medium resulted in partial desensitization to anti-IgE-induced histamine release. Pretreatment of basophils with pH 3.9 lactic acid buffer, which dissociates basophil IgE from its receptors, markedly reduced the capacity of basophils to release histamine in response to macrophage factor. Basophils that were incubated with IgE myeloma (but not with IgG) after lactic acid treatment partially or completely regained their capacity to release histamine in response to macrophage factor. Fluid-phase IgE myeloma (15 micrograms/ml) (but not IgG) inhibited basophil histamine release induced by two macrophage-derived supernatants, whereas IgE myeloma (200 micrograms/ml) did not inhibit release due to other supernatants. IgE-affinity columns removed the histamine-releasing activity of five macrophage-derived supernatants, and IgG-affinity columns had similar effects. However, neither affinity column removed the histamine-releasing activity of three other macrophage-derived supernatants. On Sephadex G-75 chromatography, nearly all of the histamine-releasing activity migrated as single peak with an apparent m.w. of 18,000. These results suggest that, although macrophage factor are heterogeneous, they are related, as they are a IgE-dependent factors that induce histamine release by interacting with cell surface IgE. These macrophage factors may be responsible for stimulation of basophil/mast cell mediator release in chronic allergic reactions.     

Clearance of lysosomal hydrolases following intravenous infusion. Kinetic and competition experiments with beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Clearance experiments with highly purified lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum β-glucuronidase and epididymal N-acetyl-β-d-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland β-glucuronidase, liver lysosomal β-glucuronidase, and liver lysosomal N-acetyl-β-d-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of β-glucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with β-glucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding.     

Characterization of murine lymphocyte IgE receptors by flow microfluorometry

A flow microfluorometric technique has been developed to analyze IgE receptors on splenic and mesenteric lymph node mononuclear cells from BALB/c mice. Our data show that 1) the binding of DIBADL cross-linked IgE dimers to IgE receptors is specific in that it is inhibited by monomeric rat and mouse IgE but not by mouse or rabbit IgG or by the monoclonal anti-Fc gamma R antibody 2.4G2, and conversely, the binding of DIBADL cross-linked IgG dimers is inhibited by monomeric IgG or 2.4G2 but not by rat or mouse IgE; 2) the binding of IgE dimers is saturable on cells from uninfected and Nippostrongylus brasiliensis (Nb)-infected mice; 3) IgE dimer binding is detectable on most splenic B lymphocytes from uninfected and Nb-infected mice, but not on T lymphocytes from uninfected mice, and on few, if any, T lymphocytes from Nb-infected mice; 4) Nb infection causes a parallel increase in the percentages of B lymphocytes and cells expressing IgE receptors and Fc gamma R; 5) Nb infection leads to a marked increase in B lymphocyte IgE receptor expression, has little if any effect on IgE receptor affinity, and causes only minor changes in Fc gamma R expression; and 6) in vivo activation of B lymphocytes by a goat antibody to mouse IgD decreases IgE receptor expression considerably, but has a minimal effect on Fc gamma R expression. Thus, there are separate receptors for IgE and IgG on murine B lymphocytes, and the effect of Nb infection or anti-IgD treatment on their expression is different.     

Binding the low affinity Fc epsilon R on B cells suppresses ongoing human IgE synthesis

Our results support the hypothesis that binding the low affinity Fc epsilon R (Fc epsilon R-II, CD23) on IgE-secreting B cells, directly suppresses IgE production. IgE production from AF-10/U266 (a human IgE plasmacytoma) decreased upon incubation with anti-IgE mAb or IgE:anti-IgE immune complexes (IgE-IC). Synthesis was suppressed a maximum of 51% with 10 micrograms/ml of IgE-IC after a 24-h incubation. Spontaneous in vitro IgE synthesis from the B cells of highly atopic individuals was also inhibited in a similar fashion. This effect was isotype specific as IgA or IgG immune complexes did not alter IgE production from AF-10 nor did IgE-IC affect IgA or IgG synthesis from lymphoblastoid cell lines making IgG (GM1500 and RPMI 8866) or IgA (GM1056). U266/AF-10 cells displayed both membrane IgE (greater than 90%) and Fc epsilon R-II (23%). To evaluate the role of these membrane proteins in the observed suppression of IgE synthesis, we treated U266/AF-10 cells with IgE-IC that bound Fc epsilon R-II but could not bind membrane IgE, as the mAb used was directed against an idiotypic determinant on the myeloma IgE (PS) used to make the IgE-IC. Suppression was maximal (greater than 50%) with these complexes at 0.1 micrograms/ml and at a 1/1 ratio of mAb anti-IgE to human myeloma IgE. When IgE-IC were used that were constructed with heat denatured IgE or F(ab')2 fragments of IgE, suppression was abrogated indicating IgE-Fc epsilon R binding was required. Neither PS IgE nor mAb 5.1 (the components of IgE-IC) alone affected IgE synthesis. Furthermore, a mAb binding directly to CD23 suppressed IgE synthesis from AF-10 up to 60%. Using limiting dilution analysis, we determined that IgE production per AF-10 cell was constant (0.9 pg/cell/24 h), independent of cell density and cells incubated with IgE-IC were uniformly suppressed. To clarify the mechanism of IgE-IC-induced suppression on AF-10 cells, we assessed both the proliferative rate and cell cycle distribution upon incubation with IgE-IC. There was no correlation between IgE production and [3H]TdR incorporation by AF-10 cells incubated with IgE-IC or anti-CD23 mAb. The distribution of cells within the cell cycle was unaffected by these treatments, with 60% of the cells in G1. These results define a direct role for the Fc epsilon R-II on B cells in the regulation of ongoing IgE synthesis.     

beta-Glucuronidase release from human monocytes induced with aggregated immunoglobulins of different classes

We studied beta-glucuronidase release from human monocytes induced with aggregated immunoglobulins of the nine different human classes and subclasses. Release was induced in a time and dose-dependent manner by all aggregated IgG subclasses. Aggregated IgA1 caused a greater beta-glucuronidase release than aggregated IgM, IgD, and IgE, but the difference was not statistically significant. Release of beta-glucuronidase was not phagocytosis dependent since inhibition of phagocytosis by cytochalasin B or dihydrocytochalasin B did not diminish enzyme release. On the contrary, cells incubated with cytochalasin B prior to addition of aggregated IgG released approximately twice as much enzyme compared to untreated controls. Enzyme release induced by latex particles, a non-Fc receptor mechanism, was decreased by cytochalasin B. Monomeric IgG1, IgG2, IgG3, and IgG4 inhibited aggregated IgG1 enzyme release in a dose-dependent manner. The ability of monomeric IgG to inhibit beta-glucuronidase release correlated with previous reports describing the binding affinities of monomeric IgG to monocytes, i.e., IgG2 was relatively ineffective compared to the other subclasses. Monomeric IgA, IgE, and pentameric IgM were unable to diminish IgG-induced enzyme release. The data indicate that normal peripheral blood monocytes express predominantly Fc receptors for IgG and that all four IgG subclasses induce the release of the lysosomal enzyme beta-glucuronidase.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.