Hepatic uptake of small-latticed immune complexes does not alter mononuclear phagocyte system function.

The hepatic and splenic uptake of circulating, small-latticed immune complexes and the effect of these complexes on the hepatic mononuclear phagocyte system (MPS) were examined in mice. The small-latticed immune complexes were prepared at fifty-fold antigen excess. The clearance from circulation and uptake by the liver and spleen of two probes of MPS function, aggregated human IgG and aggregated mouse albumin, were quantified. The hepatic uptake of a dose of small-latticed complexes, containing 5 mg of antibodies, at 1 hr was comparable with the uptake of a similar dose of complexes that contained large-latticed complexes. At later time points, the hepatic uptake of the small complexes was significantly less than that of the larger complexes. The splenic uptake of the small-latticed complexes was less at all time points. Doses of the small-latticed complexes, ranging from 1 to 5 mg antibody in the complexes, produced no significant inhibition of the clearance or organ uptake of the MPS probes when administered 1 hr after the preload injections. In contrast, large-latticed complexes produced a dose-dependent delay in clearance due to a decreased hepatic uptake of the probes. These observations showed that small-latticed immune complexes were ineffectively removed by the hepatic MPS and that the presence of large quantities of small-latticed complexes in circulation did not alter MPS function.     

Demonstration of hepatic Fc receptor function in vivo

We describe an investigation of the Fc-receptor dependent function of the hepatic component of the mononuclear phagocyte system (MPS) in rats. Erythrocytes sensitized with the rat IgG2b monoclonal antibody (JY1/98) were cleared by the liver in decomplemented splenectomized rats. This immune clearance was Fc-receptor dependent since it was effectively inhibited by immune complexes of bovine serum albumin (BSA) and rabbit-anti-BSA antibody formed either in vivo or in vitro. Immune complexes formed with F (ab')2 fragment of the rabbit anti-BSA antibody had no effect. Heat-aggregated human gamma globulin was virtually without any competitive activity in the Fc-mediated clearance system. Immune complexes inhibited the hepatic clearance of antibody-sensitized erythrocytes but did not have a significant effect on the early rapid removal of erythrocytes pre-coated with antibody and complement.     

Studies of mononuclear phagocyte function in the rat. I. Saturation and recovery following intravenous administration of soluble human serum albumin (HSA)--anti-HSA complexes.

We have attempted to demonstrate saturation of the mononuclear phagocyte system (MPS) in the Long Evans rat following intravenous administration of increasing doses of soluble HSA--125I-anti-HSA complexes. The fate of large (greater than 11S) complexes was followed by sucrose density gradient ultracentrifugation of serial serum samples in rats receiving 0.005-0.16 mg anti-HSA/g body weight. Administration of complexes provoked a rapid and profound increase in vascular permeability. Under these conditions no steady state clearance velocity for greater than 11S complexes could be established. The quantity of greater than 11S complexes removed from the circulation in the 1st hr never became independent of the initial dose. Specific immune complex uptake by the liver reached a maximum in rats receiving 0.09 mg anti-HSA/g body weight. Above this dose specific uptake decreased. Clearance of a tracer dose of complexes in rats pre-loaded with complexes containing 0.06 mg anti-HSA/g body weight was delayed for more than 3 hr. This was cautiously interpreted to indicate the period required for MPS recovery. The pattern of immune complex clearance in the context of marked changes in vascular permeability raised the possibility that maximum uptake was determined not by saturation of the mononuclear phagocyte system but by impaired hepatic perfusion.     

Studies in cobra venom factor treated rats of antibody coated erythrocyte clearance by the spleen: differential influence of red blood cell antigen number on the inhibitory effects of immune complexes on Fc dependent clearance

The splenic component of the mononuclear phagocyte system (MPS) was investigated in decomplemented rats by determining the clearance from the blood of erythrocytes coated with a monoclonal antibody (R3/13). The infusion of immune complexes (IC), prepared at 10-fold antigen excess, at an appropriate time during the erythrocyte clearance produced a significant increase in the T1/2 of the antibody coated cells. Immune complexes formed with the F(ab')2 fragment of the rabbit antibody did not have any significant effect. A positive correlation was seen between the dose of immune complex infused and the degree of inhibition of erythrocyte clearance. The influence of red cell antigen number on the behaviour of erythrocytes sensitized with R3/13 was studied by comparing the clearance of DA and (DA X PVG) F1 erythrocytes. F1 erythrocytes, with only half the number of specific antigens on their surface that bind R3/13 antibody were cleared much more slowly (82 +/- 2.6 min, mean +/- s.e.) by the spleen than the DA erythrocytes (44 +/- 1.5 min P less than 0.001). Both cell suspensions were equally susceptible to inhibition by soluble IC. These studies show that the number of specific antigens on the red cell surface influences the rate at which sensitized cells are removed by splenic macrophage Fc receptors but not their susceptibility to inhibition by IC. Our results draw attention to a major defect in the use of autologous erythrocytes coated with anti-rhesus (D) immunoglobulin to assess macrophage Fc receptor function in man.     

Prolonged circulation of immune complexes due to various altered immune functions contributes to nephritis in MRL/lpr mice.

To gain some insight into the pathogenesis of proliferative lupus nephritis in MRL/lpr mice we investigated the kinetics of removal of immune complexes from the circulation, the carrier state of blood cells, the uptake of complexes by the mononuclear phagocyte system, and the localization of complexes in kidneys. In nephritic MRL/lpr mice challenged with a subsaturating dose of radiolabelled complexes (2.5 mg bovine serum albumin-anti-bovine serum albumin) liver uptake was profoundly decreased, removal of circulating complexes was delayed, and 12-h kidney localization of complexes was enhanced 7.3-fold, in comparison to control mice. The findings were not encumbered by differences in complement concentration and most likely are attributable to various altered immune functions: spontaneous polyclonal activation of B cells, enhanced production of endogenous immune complexes, delayed removal of complexes from the circulation, and decreased uptake of complexes by the mononuclear phagocyte system. In concert, such altered functions contribute to prolonged circulation of complexes to result in their enhanced deposition in the microcirculation.     

Effect of preformed immune complexes on the clearance and tissue localization of single-stranded DNA in mice.

Recent studies have shown that DNA is cleared from the circulation extremely rapidly by the liver, and that normal individuals have low or immeasurable levels of circulating DNA. In some patients with SLE and in NZB/W mice, however, significant amounts of free DNA as well as DNA-anti-DNA immune complexes have been found in the circulation, suggesting a possible defect in DNA clearance in these conditions. To delineate factors which might contribute to the persistence of DNA in the circulation, we have assessed the effects of immune complexes on the clearance of single stranded DNA in normal C57Bl/6J mice. HSA-anti-HSA immune complexes at five-fold antigen excess were injected intravenously and after a variable, the clearance of single-stranded DNA was determined. Clearance of all doses of DNA was markedly suppressed 6 to 12 hr after the administration of immune complexes and returned to normal by 24 hr. Immune complexes decreased DNA clearance by blocking the hepatic uptake of DNA without altering the distribution of DNA to other organs. Histology and studies on the effect of immune complexes on the clearance of bromosulphophthalein (BSP) and sulphur colloid suggest that immune complexes affect DNA clearance by altering hepatic blood flow. The results obtained in this study suggest that circulating immune complexes in patients with SLE or in other conditions may suppress normal DNA clearance, and thereby contribute to the persistence of DNA in the circulation.     

Fc gamma receptor-dependent clearance is enhanced following lipopolysaccharide in vivo treatment.

Lipopolysaccharides (LPS) occupy centre stage in the pathogenesis of gram-negative sepsis. Although LPS are potent stimulators of the mononuclear phagocyte system (MPS), their effects on immune complex (IC)-specific clearance have not yet been reported. In order to evaluate this issue, we examined the MPS function after LPS treatment by measuring intravascular removal rate of syngeneic erythrocytes sensitized with specific immunoglobulin G (IgG) (EA). Our findings showed that LPS, directly or through the release of endogenous cytokines, enhance Fc gamma receptor (Fc gamma R)-dependent clearance. The EA uptake by liver, spleen and bone marrow was significantly increased leading to an effective clearance of immune complexes. Splenic antibody-dependent cellular cytotoxicity (ADCC), an in vitro indicator of Fc gamma R functionality, was also increased after in vivo LPS treatment. However, cytometric studies showed that endotoxin did not modify Fc gamma R expression on splenocytes, but markedly enhanced the expression of CD11b/CD18 (Mac-1), an adhesion molecule closely related to Fc gamma R activity. We conclude that LPS enhance Fc gamma R-dependent effector functions and suggest that this effect is mediated through alterations in adhesion molecules.     

Studies in the rat of antibody-coated and N-ethylmaleimide-treated erythrocyte clearance by the spleen. II. Effects of immune complex infusion.

The effects of immune complex infusion on the clearance of antibody (R3/13)-coated and NEM-treated rat erythrocytes by the splenic component of the rat mononuclear phagocyte system (MPS) were investigated. Equivalence complexes of BSA-anti-BSA produced a significant delay in the clearance of the NEM cells (pre-infusion T1/2 19.7 +/- 3.9 min, post-infusion T1/2 26.5 +/- 3.8 min, mean +/- SE, n = 6, P less than 0.01), but this effect could be abolished by agents that prevented the changes in the splenic blood flow that followed complement activation. The immune complexes formed in 10-fold antigen excess (mean size 15S) did not delay the clearance of the NEM cells. Clearance of R3/13-coated cells was impaired by the infusion of immune complexes prepared at equivalence. 10-fold antigen excess or complexes prepared with F(ab')2 fragments of rabbit anti-BSA antibody. The inhibition of red cell clearance was independent of changes in blood flow, but the degree of inhibition produced did not correlate well with the dose of immune complex injected.     

Effect of soluble immune complexes of Fc and C3 receptor-dependent phagocytosis by human monocytes.

The capacity was studied of bovine serum albumin (BSA) rabbit anti-BSA and ovalbumin (OA) rabbit anti-OA immune complexes of different composition to inhibit the Fc receptor-dependent adherence and phagocytosis of sensitized sheep red blood cells by human monocytes. Parallel experiments were performed on the ability of the immune but complement-reacted complexes to inhibit the C3 receptor-dependent phagocytosis of C3-coated yeast particles. The extent of inhibition of both the Fc and C3 receptor-dependent phagocytosis was proportional to the antibody avidity of the complex used. Immune complexes made at equivalence and at moderate antibody excess markedly inhibited both types of phagocytosis, whereas those made at moderate antigen excess had only a weak inhibitory effect. These findings can be explained by the correlation between the Fc receptor-binding and complement-activating capacities of immune complexes of different composition. An alternative explanation, however, is also discussed.     

Modulation of mononuclear phagocyte system function and circulating immune complexes by lyophilized concentrates in patients with classic hemophilia

Infusions of lyophilized antihemophilic factor concentrates in patients with hemophilia were found to affect both circulating immune complexes and in vivo mononuclear phagocyte system function. Following infusion nine patients had simultaneous assessment of serial immune complex levels and mononuclear phagocyte system clearance of IgG-sensitized autologous erythrocytes relative to a previously established baseline. Nine patients also had a separate second sequence, and two a third sequence, of serial immune complex measurements in relation to infusions. The net change in immune complexes over the 2- to 4-hr interval following infusion was consistent in 10 of 11 study pairs (P less than 0.01) despite different antihemophilic factor preparations for each study and different individual patient responses. This change could not be explained by immunochemical rearrangement in infusate and serum since in vitro mixing experiments showed no relationship to in vivo results. Change in mononuclear phagocyte system function showed a strong correlation with change in complexes (r = 0.70; P less than 0.05). It is suggested that infusions of antihemophilic factor can modulate the mononuclear phagocyte system which in turn alters immune complex levels. Both of these effects could potentially influence immune regulation which has been shown to be abnormal in other hemophiliac patient groups.     

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.     

Mechanism of localization of immune complexes in NZB/W mice with early nephritis

We investigated the pathogenesis of mesangial proliferative lupus nephritis in NZB/W mice under conditions that allowed us to examine removal of immune complexes from the circulation, uptake by the mononuclear phagocyte system, and localization in kidney tissue. These studies were performed at a time when variables such as the quantity of endogenous immune complexes, complement concentration, and carrier state of blood cells (platelets) were controlled. NZB/W mice and C57BL/6 (control) mice showed comparable kinetics for removal of a subsaturating dose of immune complexes (2.5 mg bovine serum albumin-antibovine serum albumin) from the circulation; additionally, the liver uptake and kidney localization of these immune complexes were comparable between NZB/W and control mice. The localization of immune complexes in the glomerular mesangium of NZB/W mice could not be attributed to enhanced production of endogenous immune complexes, to decreased removal of immune complexes from the circulation, to impaired uptake by the liver, or to complement concentration and carrier state of blood cells. It appears, by exclusion, that mesangial deposits of immunoreactants in early lupus nephritis may result from interaction of antibodies with antigens in mesangia.     

Insoluble immune complex-stimulated neutrophil leukotriene B4 production is dependent on Fc gamma RII and Fc gamma RIII and independent of pertussis toxin-sensitive signal transduction pathways.

Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis of LTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc gamma RII or Fc gamma RIII receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble ICs. However pertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc gamma RII and Fc gamma RIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc gamma RII and Fc gamma RIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA. These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils.     

Effect of carrageenan on activity of the mononuclear phagocyte system in the mouse

Injection of 5 mg carrageenan caused hepatosplenomegaly and thymic involution and resulted in temporary blockade of the mononuclear phagocyte system (MPS). Impaired MPS activity was shown by decreased hepatic phagocytosis of i.v. injected colloidal carbon and 51Cr-labelled sheep red blood cells (SRBC). Depression of Kupffer cell activity was dependent on carrageenan dose, route of administration and interval between carrageenan and particle injection. Reduction in hepatic uptake of SRBC was accompanied by increased localization of these cells within the spleen.     

Effect of carrageenan on activity of the mononuclear phagocyte system in the mouse.

Injection of 5 mg carrageenan caused hepatosplenomegaly and thymic involution and resulted in temporary blockade of the mononuclear phagocyte system (MPS). Impaired MPS activity was shown by decreased hepatic phagocytosis of i.v. injected colloidal carbon and 51Cr-labelled sheep red blood cells (SRBC). Depression of Kupffer cell activity was dependent on carrageenan dose, route of administration and interval between carrageenan and particle injection. Reduction in hepatic uptake of SRBC was accompanied by increased localization of these cells within the spleen.     

Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes.

Complement receptor 1 (CR1) on primate red blood cells (RBC) binds most complement-fixing immune complexes in the circulation. It has been postulated that by binding them, RBC keep immune complexes in the intravascular space and deliver them to the tissue macrophages of the mononuclear phagocyte system. We have developed an in vitro model to study the transfer of RBC-bound immune complexes (heat-aggregated IgG and DNA-anti-DNA) to phagocytic cells (human monocytes). Transfer of immune complexes from RBC to monocytes occurred significantly more rapidly than monocyte uptake of the same immune complexes from solution. In the transfer process, complex-bearing RBC were not bound or sequestered by the monocytes. To define the monocyte receptors involved in binding immune complexes from the RBC surface, monocyte receptors were blocked with MoAbs (anti-CR1, anti-FcRII) or EDTA (to block CR3). Monocyte binding of immune complexes primarily used CR1 with a small contribution from FcRII, and with little or no contribution from CR3 and FcRI. Uptake of immune complexes from solution employed the same monocyte receptors as binding of complexes from the RBC surface. Immune complexes in solution bound to RBC and to monocytes with equally high avidity (approximately 1 x 10(11) l/M), but monocytes expressed a 15-20-fold greater number of immune complex binding sites. We propose that immune complexes distribute between RBC and monocytes according to the binding capacity of these cells, such that at equal or high RBC/monocyte ratios as would be seen in the circulation immune complexes bind to RBC, but at low RBC/monocyte ratios (as would be seen in the sinusoidal circulation of the liver and spleen), most immune complexes bind to monocytes. To define the pathway by which immune complexes move from RBC to monocytes, their release from RBC CR1 was examined. Under various conditions, the dissociation rate was extremely slow, and did not increase with the addition of monocyte supernatants. To examine whether factor I-mediated processing of immune complexes enhances binding of immune complexes to monocytes, RBC-bound complexes were released with factor I, and binding of these 'processed' immune complexes to monocytes was examined. Monocyte binding of these processed immune complexes was slower than of control ones; furthermore, performance of transfer experiments at 4 degrees C, which significantly shows enzymatic processes, did not decrease the rate of immune complex transfer from RBC to monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of immune complexes using a solid-phase C1q polystyrene ball assay

A polystyrene ball C1q solid-phase assay (PSB C1q SPA) has been developed for quantitating immune complexes in human serum. While similar to the previously reported solid-phase C1q assays in principle, the use of polystyrene balls with a specular finish has resulted in assay with significantly improved accuracy, sensitivity and reproducibility. The sensitivity of the assay based on the amount of AHGG bound per μg C1q added was approximately 12-fold higher in the PSB assay compared to the polystyrene tube solid-phase C1q method.

In order to correct for variable background contributions in different samples, values obtained with heat-inactivated C1q were subtracted from each experimetal result. Reproducibility studies yielded a coefficient of variation (CV) of 10 and 4% in day-to-day assays using 25 μg and 100 μg AHGG/ml normal serum, respectively compared to 11–15% for the tube technique. Within run measurements gave a CV of 37 and 20% at the low and high levels of AHGG.

Aggregated human gamma-globulin (AHGG) was used as a model immune complex and when chromatographed on Biogel A-15m yielded major fractions at 15,000,000 and 150,000 daltons. Maximum binding of AHGG to PSB C1q occurred with aggregates greater than 15,000,000 daltons.

Optimum binding of human albumin-anti-albumin complexes in the PSB C1q SPA occurred at a molar ratio of 1:1.5. The size distribution of this complex active in the assay determined by sucrose density gradients was 14–32 S with peaks at 21 and 27 S.

A normal range of immune complexes was determined as 15±8μg AHGG equivalents (±2 S.D., n = 65). Approximately 70% of rheumatoid arthritis, linear scleroderma, vasculitis, Sjögren's and glomerulonephritis and 40% of SLE patients were above 2 S.D. of normal. SLE patients demonstrated elevations in immune complexes only during periods of increased disease severity. The assay was a useful monitor of plasmapheresis in an SLE patient, showing decreases in immune complexes after each plasmapheresis. DNA did not interfere with AHGG binding whereas lipemia prevented detection of added AHGG.     

A new enzymatic assay for evaluating the clearance of immune complexes from the circulation of mice.

A new photometric in vivo enzymatic immune complex clearance (EIC) assay was developed in a homologous system using glucose oxidase-anti-glucose oxidase complexes (GAG) as a model of immune complexes. Chromatographically purified GAG was injected into mouse tail veins and at intervals thereafter the enzyme activities of GAG remaining in the circulation were estimated. The GAG were cleared in a size dependent manner and were stable, being eluted as the same discrete peaks on HPLC size-exclusion chromatography both before and after injection into mice. The complement consuming activity of the GAG was weak, and depletion of complement components with cobra-venom factor did not alter clearance of the GAG from the circulation, whereas pretreatment of aggregated mouse gamma globulin suppressed the clearance rate. These results suggested that most of the GAG were not cleared via complement receptors but via FcR. Normal clearance rates were significantly changed by administration of immunomodulators such as carrageenan or LPS. Intravenous administration of GAG at a dose 50 times higher than normal caused no deaths suggesting that the complexes were of low toxicity. The enzymatic method presented should be of value for measuring the function of the mononuclear phagocytic system with respect to immune complex clearance. It provides a rapid and sensitive alternative assay which avoids using radioisotopes.     

Effect of cyclophosphamide on the clearance of IgG-sensitized red cells in mice

The effects of cyclophosphamide (Cy) on the clearance of IgG-sensitized erythrocytes (EA) were examined. The results clearly indicate that Cy treatment enhances the capacity of the mononuclear phagocytic system to remove antibody-coated cells from the circulation in normal and decomplemented mice. The enhanced rate of clearance is the consequence of an increased uptake of EA by the liver and spleen. We explored the possibility that the enhancement of Fc receptor-mediated clearance might be an important effect to be taken into account in the search for a more effective therapy of immune complexes diseases.     

Complement-mediated delay in immune complex clearance from the blood owing to reduced deposition outside the reticuloendothelial system

The effect of serum complement upon the blood clearance rate and tissue distribution of intravenously injected small immune complexes formed between 125-labelled dinitrophenyl (DNP)-conjugated human serum albumin and rabbit IgG-anti-DNP antibodies was studied in mice. Immune complexes formed in the absence of complement (i.e. in phosphate-buffered saline or in the presence of complement-depleted serum from mice treated with cobra venom factor) were cleared from the blood at a higher rate than was the antigen injected alone. In contrast, immune complexes formed in the presence of fresh normal mouse serum were cleared from the blood at the same rate as was the native antigen. Immune complexes formed in the presence of complement were eliminated mainly by the liver. The complexes formed in the absence of complement also became located to the liver to approximately the same extent, but an even larger proportion of these immune complexes was deposited outside the reticuloendothelial system. It is concluded that serum complement may inhibit IgG-mediated clearance of antigen, probably by increasing the solubility of the complexes, thereby reducing the risk of deposition outside the reticuloendothelial system.