Binding and catabolism of aggregated immunoglobulins bearing C3b or iC3b by U937 cells.

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CR1 and CR3 of human monocyte cell line U937 on the complement-mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgG to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgG to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes.     

Complement-dependent inhibition of degradation of soluble immune complexes and immunoglobulin aggregates by thioglycollate-stimulated peritoneal macrophages.

Previous studies have shown that the degradation of soluble immune complexes or aggregates (AIgG) by normal peritoneal macrophages can be enhanced by complement. The enhancement of degradation was shown to be at least in part dependent on the number of C3b molecules bound per complex. The present investigations indicate that the enhanced degradation is not found with thioglycollate-stimulated macrophages, and that at high concentrations of complement, inhibition may even occur. The Fc receptor-mediated degradation of soluble immune complexes and AIgG by stimulated macrophages was at least twice as high as that by normal macrophages. This increase was compatible with the increased number of Fc receptors on the stimulated macrophages. The inhibitory effect of high concentrations of serum, as a complement source, on the degradation of AIgG was dependent on the number of C3b molecules bound per AIgG. Although there was also a two-fold increase in the number of C3b receptor sites on the stimulated macrophages, more than 11 C3b molecules per AIgG40 caused significant inhibition of degradation. This phenomenon may be dependent on shielding of Fc-Fc receptor interaction by varying numbers of C3b molecules per complex.     

The role of cellular Fc and C3 receptors on the complement-dependent degradation of stable soluble immunoglobulin aggregates by normal and trypsin-treated peritoneal macrophages.

The experiments described in this paper were designed to determine the degradation of soluble IgG aggregates (AIgG) bearing C3b by guinea-pig peritoneal macrophages with different numbers of functional C3b receptors. The number of functional Fc receptors for AIgG containing 40 molecules of IgG per aggregate (AIgG40) were kept constant during these experiments. By increasing the concentration of trypsin for the treatment of normal peritoneal macrophages a dose-dependent inactivation of C3b receptors was achieved as determined by the binding of tetrameric [125I]-C3b([125I]-AC3b). Normal peritoneal macrophages bound 148,500 AIgG40 and 60,300 AC3b per cell. Treatment of the macrophages for 15 min at 37 degrees with incremental amounts of trypsin from 60 to 1000 micrograms/ml did not affect the binding of AIgG40 to macrophages but caused a dose-related loss of up to 95% of AC3b binding, indicating functional inactivation of C3b receptors. Degradation of trichloroacetic acid non-precipitable protein of [125I]-AIgG40 by 10(6) macrophages was 38 +/- 6% in medium alone and 60 +/- 8% in the presence of 10% fresh guinea-pig serum (NGPS) after 60 min at 37 degrees. The inactivation of 25%, 50%, 72% and 94% of C3b receptors by trypsin did not affect the degradation of AIgG40 in medium alone, but decreased degradation from 69% to 58%, 47%, 32% and 13% of AIgG40 respectively, in the presence of 10% GPS. Macrophages (10(6)) in medium alone degraded 40%, 47%, 65% and 68% of AIgG40 bearing 0, 5, 10, 16 and 20 C3b molecules per aggregate, indicating a dose-dependent enhancement of degradation by bound C3b; conversely, inactivation of 95% of C3 receptor on the macrophages resulted in 39%, 30%, 12%, 5% and 6% degradation of these AIgG40-C3b, indicating a dose-related inhibition by aggregate-bound C3b in the absence of cellular C3 receptor. These experiments stress the importance of Fc and C3b receptor co-operation and immune complex bound C3b.     

Degradation of Soluble Immunoglobulin Aggregates in Vitro by Monocytes from Normal Subjects and from Patients with Systemic Lupus Erythematosus

The capacity of human peripheral monocytes to degrade soluble immunoglobulin (IgG) aggregates (AIgG) was studied in vitro. Under serum-free conditions peripheral monocytes from normal donors were able to degrade soluble AIgG in a linear and time-dependent fashion. Addition of fresh human or fresh guinea-pig serum to the incubation mixtures caused a marked increase in degradation of the amount of soluble AIgG available. The stimulatory effect of fresh serum was complement-mediated, because it was abolished by heat treatment of the serum and was not seen when C4- or C3-deficient sera were tested. Functional inactivation of C3 receptors on the phagocytes by trypsin also abolished the complement-mediated stimulation, suggesting cooperation between Fc and C3 receptor in degradation of soluble AIgG. No significant differences were found between monocytes from normal donors and those from patients with systemic lupus erythematosus, as far as degradation is concerned in the presence of complement.     

Fc- and complement receptor-dependent degradation of soluble immune complexes and stable immunoglobulin aggregates by guinea pig monocytes, peritoneal macrophages, and Kupffer cells

The degradation of soluble immune complexes (ICx) and stable soluble immunoglobulin aggregates was studied in vitro. To obtain insight into the capacity of phagocytes from different organs to degrade soluble ICx we studied monocytes, peritoneal macrophages, and Kupffer cells. Peritoneal macrophages and Kupffer cells degrade similar amounts of aggregated guinea pig IgG2 (AIgG) and ICx per cell. Monocytes were at least ten times less effective than peritoneal macrophages and Kupffer cells. The presence of normal guinea pig serum as a source of complement enhanced the degradation of ICx and AIgG by peritoneal macrophages and monocytes. There was, however, a diminished degradation of ICx and AIgG by freshly isolated Kupffer cells in the presence of complement. After culturing of the Kupffer cells for 40 hours, there was an increase in the density of C3b receptors and a concomitant reversal of inhibition of AIgG degradation in the presence of complement. It can be concluded from the present experiments that the capacities of peritoneal macrophages and Kupffer cells to degrade soluble ICx and AIgG are comparable and that monocytes are much less active than peritoneal macrophages and Kupffer cells.     

Acidic pH increases the avidity of FcgammaR for immune complexes

The interaction of immunoglobulin G (IgG) antibodies with FcgammaR constitutes a critical mechanism through which IgG antibody effector functions are mediated. In the current work we have examined whether human neutrophil FcgammaR exhibit pH dependence in their association with IgG. Binding assays were performed in culture medium adjusted to different pH values. It was found that the binding of either heat-aggregated human IgG (AIgG), soluble immune complexes (sIC) or IgG-coated erythrocytes (IgG-E) was markedly higher at pH 6.5 than at pH 7.3. This effect was not observed when saturation of FcgammaR was achieved, suggesting that acidic pH increases the avidity of FcgammaR for IC without modifying the total binding capacity. Similar results were observed for the binding of AIgG to either monocytes, natural killer (NK) or K562 cells, suggesting that acidic pH increases the avidity of both, FcgammaRII and FcgammaRIII. Additional experiments were performed to analyse whether the binding of IgG to FcgammaRI also showed pH dependence. To this aim, we employed interferon-gamma-treated human neutrophils and mouse inflammatory macrophages, previously incubated with blocking antibodies directed to FcgammaRII and FcgammaRIII. Acidic pH did not enhance the binding of AIgG nor monomeric IgG under these experimental conditions. Further studies are required to determine whether the enhancement of FcgammaR avidity for IC could be attributed to titration of histidine(s) residues on the Fc fragment of IgG.     

Effects of soluble aggregates of IgG on the binding, uptake and degradation of the C1q subcomponent of complement by adherent guinea pig peritoneal macrophages

Earlier studies have indicated that C1q, the first subcomponent of complement component C1, is bound to lymphocytes via specific C1q receptor sites. We have recently shown that adherent guinea pig peritoneal exudate macrophages express specific receptors for C1q (Veerhuis, R. et al., Immunology 1985. 54: 801). The present studies were performed to determine whether binding of 125I-labeled human C1q (125I-C1qhu) to adherent guinea pig peritoneal exudate macrophages would also result in ingestion and subsequent degradation of 125I-C1qhu. The binding of 125I-C1qhu to adherent peritoneal macrophages at 4 degrees C is inhibited fully not only by C1qhu and guinea pig C1q (C1qgp) but also by pepsin fragments of C1qhu. The amount of trichloroacetic acid nonprecipitable radioactivity that appeared in the supernatant was used as a measure for the degradation of 125I-C1qhu. 125I-C1qhu is degraded initially into fragments of 25 kDa, after which it is degraded further into small molecular weight peptides. Ingestion of 125I-C1q by the macrophages occurs before the 125I-C1q is degraded. In the presence of limited amounts of soluble aggregates of guinea pig IgG2 (AIgG), a known activator of C1, part of the C1q is bound to the AIgG and all of the AIgG in turn is bound to the cellular Fc receptors leading to an enhanced binding of 125I-C1q to the cells, a binding that was maximal at near equimolar concentrations of 125I-C1qhu and 131I-AIgG. In the presence of a 30-fold excess of AIgG, however, only a small percentage of the AIgG binds to cellular Fc receptors and the interaction of C1q with its receptor is decreased due to competitive inhibition. The results presented in this report thus suggest that free C1q may be eliminated by specific interaction with C1q receptors present on circulating and tissue phagocytoses and, in addition, that in the presence of immune complexes modulation of elimination of C1q may be encountered.     

Neutrophil Fcγ and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fcγ receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fcγ and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

Kupffer cell depletion in vivo results in preferential elimination of IgG aggregates and immune complexes via specific Fc receptors on rat liver endothelial cells.

In the present study we have investigated the clearance kinetics and tissue distribution of monomeric (m) IgG and soluble aggregates of IgG (AIgG) and immune complexes (IC) in normal and Kupffer cell (KC) depleted rats. In normal rats, clearance of mIgG occurred in a biphasic manner with a first half-life (T1/2) (T1) of 36.3 +/- 6.3 min and a second T1/2 (T2) of 168.4 +/- 4.7 min. AIgG composed of 20-27 IgG molecules per aggregate were cleared significantly faster than mIgG with a T1 of 2.5 +/- 0.1 min and a T2 of 32.5 +/- 5.6 min. KC depletion did not have a significant effect on the clearance rate of mIgG (T1: 33.4 +/- 8.9 min; T2; 159.5 +/- 12.5 min), while clearance of AIgG was delayed significantly with T1 4.8 +/- 0.7 min and T2 41.2 +/- 3.2 min. Eight minutes after injection, 77% of AIgG was found in the liver in normal rats while 62% was found in the liver of KC-depleted rats. Double immunofluorescence studies indicated that AIgG in the liver was associated with KC and endothelial cells (EC) in normal rats. In KC-depleted rats, AIgG was strongly associated with EC. A similar staining pattern was observed when IgG-immune IC were administered. The clearance of AIgG in KC-depleted rats was inhibited fully by pre-administration of high concentrations of IgG but not by pretreatment with IgA. asialofetuin (ASFe) or ovalbumin (OVA). Aggregated F(ab')2IgG was cleared with a comparable rate to mIgG from the circulation, again suggesting Fc gamma receptor-mediated elimination of AIgG by EC. There was a reduced degradation of AIgG in rats depleted of KC as compared with normal rats. These data suggest binding and degradation of AIgG by EC in vivo.     

Shedding and synthesis de novo of Fc and C3b receptors by cultured guinea-pig macrophages.

Resident macrophages freshly obtained from the peritoneal cavity of guinea-pigs were demonstrated to form a higher percentage of Fc and C3b rosettes than elicited macrophages when low concentrations of IgG and IgM-C3b were used to sensitize ox red blood cells (ORBC) in rosette assays. Culture of the total resident and elicited macrophages for 6 h at 37 degrees C resulted in a decrease of Fc and C3b rosette-forming cells, the loss of Fc receptor-bearing cells by resident macrophages only being apparent when using a sub-optimal concentration of sensitizing IgG. After 24 h incubation the percentages of Fc and C3b rosettes returned to their initial values. In contrast, there was no decline in the percentage of Fc and C3b rosettes formed by the adherent population of resident and elicited macrophages cultured for 6 h. However, extending the incubation of the adherent macrophage to 24 h produced an increase of Fc receptor-positive cells and a dramatic decrease of C3b receptor-positive cells. Culture supernatants of the total macrophage population that had been incubated for 6 h inhibited Fc and C3b rosette formation by freshly obtained elicited macrophages. These results, together with the demonstration that treatment of the total macrophage population with cycloheximide led to an inhibition of Fc and C3b receptor expression after 24 h culture, suggest that the Fc and C3b receptors of guinea-pig macrophages are shed and synthesized de novo during short-term culture. This system could be applied to the study in vitro of soluble immunoregulatory mediators on macrophage functions which are dependent on the expression of Fc and C3b receptors.     

Complement enhances the elimination of soluble aggregates of IgG by rat liver endothelial cells in vivo

In the present study, we have investigated the role of complement (C) and possible C receptors present on rat liver endothelial cells (EC) in the clearance and tissue distribution of soluble aggregates of IgG (AIgG). To study the effect of elimination of AIgG by EC in vivo, Kupffer cell (KC)-depleted rats were used, with or without an intact C system (These rats will be referred to throughout this report as EC-rats.) In EC-rats with an intact C system, clearance of AIgG (2000-3000 kDa, 20-27 IgG molecules/aggregate) occurred in a biphasic manner with a first T½(T1)of 9.4 ± 2.3 min and a second T½(T2) of 44.7 ± 16.0 min. In EC-rats without an intact C system [cobra venom factor (COVF)-treated group], clearance of AIgG was significantly delayed with a T1 of 25.3 ± 9.9 min (p 0.005) and aT2 of 124.5 ± 18.4 min (p 0.001). There were less degradation products of AIgG in the circulation in EC-rats treated with COVF as compared to EC-rats with an intact C system. Eight minutes after injection, 27.5 ± 11.6% of the injected AIgG was found in the livers of EC-rats while 15.1 ± 3.2% was found in the livers of the COVF-treated group. Double immunofluorescence studies indicated that AIgG in the liver was associated with EC in the rats with an intact C system. Clear deposits of C3 and lesser amounts of Clq accompanied the deposition of AIgG. In COVF-treated EC-rats, AIgG together with Clq was also associated with EC but no detectable C3 was seen. These data suggest clearance of AIgG via Fc and C receptors present on EC in vivo.     

Human neutrophil Fc receptor-mediated adhesion under flow: a hollow fibre model of intravascular arrest.

Human polymorphonuclear cells (PMN) were found to adhere to a novel model of blood vessel wall-associated IgG. The internal surfaces of cellulose acetate hollow fibres, of comparable internal diameter to small blood vessels, were coated with normal serum human IgG, heat-aggregated IgG (HAIgG), laminin or fibrinogen. Under conditions of flow mimicking those in a small vessel, PMN were found to adhere markedly only to immunoglobulin-coated fibres. Arrest on HAIgG was inhibited by excess soluble IgG but not by bovine serum albumin (BSA), demonstrating that the adhesion was IgG-specific and presumably mediated by Fc gamma R on the PMN surface. Pre-adsorption of serum components onto HAIgG-coated fibres enhanced PMN arrest, due most probably to fixation of complement components by immobilized HAIgG, resulting in additional potential to entrap PMN via complement receptors such as CR3. Treatment of PMN with the regulatory neuropeptide substance P also enhanced adhesion to HAIgG-coated fibres and caused increased surface expression of Fc gamma RI, Fc gamma RII and Fc gamma RIII. A mouse cell line derived from L cells, hR4C6, stably transfected with human Fc gamma RII, was found to adhere under flow to HAIgG-coated fibres, whilst untransfected parent L cells did not. This adhesion was similarly inhibited by excess soluble IgG, confirming the capability of Fc gamma R to mediate cell arrest. The study strongly suggests that Fc gamma R may play an important role in intravascular PMN arrest and we speculate that in inflammatory diseases PMN may adhere via Fc gamma R to immobilized immunoglobulin on the vascular endothelium, with subsequent degranulation and tissue damage.     

Abnormal clearance of soluble aggregates of human immunoglobulin G in patients with systemic lupus erythematosus.

In the present study, we tested mononuclear phagocyte system function in nine healthy controls and 15 SLE patients with complement activating 123I-labelled aggregates of human IgG (AIgG). Clearance half-time of AIgG was 26 +/- 8 min in controls, compared to 58 +/- 27 min in patients (P less than 0.005). Binding of AIgG to erythrocytes was significantly lower in patients, 9.3 +/- 8.1 vs 24 +/- 20% (P less than 0.05). The increase of C3a-levels in plasma was significantly lower in patients than in controls (P less than 0.05 at 3 and 8 min), suggesting less complement activation. Liver and spleen uptake of 123I-AIgG was measured with a gamma camera and expressed as liver/spleen uptake ratios. In patients, the liver/spleen uptake ratios were significantly higher than in controls at 15 min, 3.8 +/- 2.0 vs 2.31 +/- 0.7 (P less than 0.05), due to less splenic uptake of AIgG. Correlations between clearance half-time or liver/spleen uptake ratios and immune complex levels or disease activity were not found. This study indicates that clearance of soluble AIgG is abnormal in patients with SLE, due to decreased splenic uptake of AIgG.     

Detection of the binding of IgG2a and IgG2b on the surface of macrophages from mice chronically infected with lactic dehydrogenase virus.

This study showed that sera from mice chronically infected with lactic dehydrogenase virus (LDV) contained virus-antibody complexes (IC). IgG2a and IgG2b, but not IgG1, IgG3, IgM or IgA, were demonstrated on the surface of macrophages from chronically infected mice. These results suggest that IC in the circulation may bind to Fc receptors for IgG2a and IgG2b on the surface of macrophages and lead to the modulation of macrophage function seen in chronically LDV-infected mice.     

Non-complement-dependent adsorption of soluble immune complexes to human red cells

Heat aggregated IgG and soluble immune complexes (IC) prepared by combining human serum albumin with rabbit anti-serum albumin and tetanus toxoid with rabbit antiserum to tetanus toxoid were shown to bind to human O+ RBC. The binding was greater for soluble IC prepared at antigen excess, and although it was usually maximal when IC were pre-opsonized, it could also be demonstrated using non-opsonized heat aggregated IgG or soluble IC prepared in the absence of complement. These observations suggest that two types of receptors may be involved in binding of soluble IC to human RBC: the classical C3b receptor, and a non-complement-dependent receptor, perhaps recognizing the Fc region of the immunoglobulin molecule exposed after heat aggregation or antigen-antibody reaction.     

Difference in clearance kinetics of particulate immune complexes and soluble aggregates of IgG in vivo

In this study the similarities and differences in kinetics and site of clearance of particulate immune complexes and soluble aggregates of IgG (AIgG) were compared in rats. The rate of clearance of both antibody-coated erythrocytes (EA) and AIgG was dependent on the number of IgG molecules per EA or AIgG. Both probes were rapidly taken up by the liver. Whereas a minor part of the AIgG returned to the circulation as TCA-soluble breakdown products, a substantial part of the injected EA returned to the circulation as intact cells after 4 min. This rebound mechanism was shown to be complement (C)-dependent. Immediately after injection the injected EA bound C3 on their surface, whereafter the number of C3-bearing cells in the circulation rapidly decreased (T1/2 7.2 min). In C-depleted rats relatively more EA were taken up by the spleen than by the liver, in comparison with EA injected into normal rats (liver/spleen ratio in C-depleted rats 0.4 +/- 0.1; in normal rats 2.1 +/- 0.4). When C3 was bound and inactivated by preincubation of EA in vitro, a similar shift from liver to spleen (liver/spleen ratio 0.4 +/- 0.1) was observed. It is suggested that when the hepatic C receptors do not participate, the splenic Fc receptors become relatively more important in the clearance of EA from the circulation.     

Soluble FcgammaRIIa inhibits rheumatoid factor binding to immune complexes

Soluble low-affinity receptors for IgG are known to inhibit immune complex (IC)-mediated inflammation, and expression by leukocytes is elevated in several inflammatory diseases. Immunoglobulin M (IgM) rheumatoid factors (RF), anti-Fc autoantibodies, are found in autoimmune diseases, such as rheumatoid arthritis (RA), as well as in normal immune responses. This study demonstrated that soluble FcgammaRIIa inhibits the interaction of rheumatoid factors with ICs. The recombinant soluble low-affinity FcgammaR, rsFcgammaRIIa, partially inhibited (30-70%) the rate of precipitation of soluble ICs by RF-positive RA sera. This required the normal interaction of FcgammaRIIa with Fc as the effect could be abrogated with the Fab fragment of the blocking mAb IV-3. Furthermore, rsFcgammaRIIa partially inhibited (40%) the binding of a monoclonal IgM RF (RF-AN) to an IC formed by IgG2 antibody binding to an antigen-coated biosensor chip. Since RF-AN has been characterized by crystallography to bind to the CH2/CH3 interface of the IgG-Fc, and leukocyte FcgammaRIIa binds to a distinct site centred on the lower hinge, this inhibition is uncompetitive. Some inhibition (15%) of staphylococcal protein A binding to IC was also observed. As soluble FcgammaRIIa disrupts Fc:Fc interactions in IgG-ICs, we propose that this alteration of the IC also reduces the accessibility of Fc portions in the IC, resulting in the partial inhibition of ligands, particularly IgM RF, which bind Fc. We propose that the high concentrations of soluble FcgammaR found during inflammation can affect the properties of ICs and their interaction with the immune system.     

Clearance of soluble aggregates of human immunoglobulin G in healthy volunteers and chimpanzees.

Using aggregates of IgG (AIgG) obtained by heat aggregation of a 16 g% human IgG solution, we sought a method for measuring the function of the mononuclear phagocyte system with a probe that bears more resemblance to soluble immune complexes than erythrocytes coated with anti-rhesus IgG (EIgG). It was found that intravenous administration of 10 micrograms AIgG/kg body weight did not cause any detectable side effects in chimpanzees. In nine healthy volunteers, a dose of 10 micrograms AIgG/kg body weight was used without any adverse reactions. AIgG is cleared from the human circulation with a t1/2 of 26 +/- 8 min (mean +/- SD). The site of clearance is predominantly the liver, as shown by an average liver spleen uptake ratio of 230:100. In whole blood obtained from the volunteers, it was found that erythrocytes bound significant amounts of AIgG, suggesting that CR1 on erythrocytes is involved in the clearance of complement activating immune complexes in humans. In five of the volunteers, clearance studies with EIgG had been done in a previous study. EIgG was cleared from the circulation with a t1/2 of 30 +/- 6.2 min (mean +/- SD). The predominant site of clearance of EIgG was the spleen. These data indicate that sensitized red blood cells are cleared from the circulation differently from soluble IgG aggregates.     

Modulation of Fc and C3b receptor expression on guinea-pig macrophages by lymphokines.

The decrease in Fc-receptor-positive cells that occurred during a 6 h incubation of resident and elicited guinea-pig macrophages was partly abrogated when lymphokines were present in the culture. When the same lymphokine preparations were tested on C3b receptor-expression they preferentially sustained the percentage of C3b rosettes formed by resident rather than elicited macrophages. This lymphokine-induced maintenance of Fc and C3b rosettes by cultured macrophages may have been due to an inhibition of receptor release or an increase in receptor synthesis. Supernatants from cultured macrophages contain shed Fc and C3b receptors which inhibit rosette formation by other macrophages. From the demonstration that culture supernatants from both lymphokine-treated and untreated macrophages significantly inhibited Fc and C3b rosette formation by freshly obtained macrophages it seems that the shedding of Fc and C3b receptors is not modified by lymphokines. The maintenance of Fc and C3b rosettes by lymphokines was inhibited by treatment of the macrophages with cycloheximide, suggesting that the lymphokine effect was due to an increase in synthesis de novo of the Fc and C3b receptors. The lymphokine-inducing antigens, BGG and PPD, and control lymphokine preparations were devoid of receptor modifying activity. The reduction in the percentage of Fc rosettes after 6 h culture appears to be due to a loss of Fc receptors for IgG1. Although lymphokines partly inhibited this effect they could not prevent the loss of these receptors following 24 h culture, unlike their action in augmenting the expression of Fc receptors for IgG2. These findings suggest that a selective enhancement of Fc receptor synthesis by lymphokines may modify the functional activities of macrophages.     

The expression of Fc and complement receptors in young, adult and aged mice.

Age-dependent changes in the expression of Fc receptors (FcR) for different isotypes of immunoglobulins and receptors for C3b, C5b and C3bi fragments of complement on the membranes of peritoneal macrophages were studied with mice of different ages. An age-related increase in expression of Fc receptors for IgM, IgE, IgA, IgG2b and IgG3, and a decrease in the expression of Fc receptors for IgG1 was observed. The expression of FcR on macrophages of donors of different ages corresponded with Fc-receptor mediated phagocytosis. The highest number of C3b-binding macrophages was found in aged mice, in contrast to low numbers of C3bi-binding macrophages at this age. The percentage of C5b-binding macrophages was lowest in adult animals. We also observed effective inhibition of binding of the C3b component of complement by preincubation of macrophages with aggregated IgG and vice versa. These observations suggest that fluctuation in expression of Fc but not C receptors may be important to the generalized changes that occur in macrophage function during development and ageing.