The glomerular mesangium: I. Kinetic studies of macromolecular uptake in normal and nephrotic rats

This study was designed to define quantitatively the function of the rat glomerular mesangium in the uptake and processing of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, AHIgG-(125)I), to relate this function to that of the general reticuloendothelial system, and to examine the effects of increased glomerular permeability to protein on the mesangial cell system.Mesangial localization of human IgG as demonstrated by immunofluorescent microscopy showed good correlation with concentrations of AHIgG-(125)I in preparations of isolated glomeruli. In normal rats the concentrations of AHIgG-(125)I in glomeruli were similar to those of lung, liver, and spleen and demonstrated a rapid decrease with increasing time intervals after aggregate administration.In rats given aminonucleoside of puromycin a marked increase in mesangial uptake of aggregates was found while studies of nephrotic lungs, liver, spleen, and blood showed no such differences. Glomerular levels of AHIgG-(125)I in aminonucleoside animals could not be correlated with the quantity of proteinuria.Nephrotic and control animals given unaggregated human IgG showed little glomerular localization by immunofluorescent microscopy; no difference in the concentration of this protein in nephrotic as compared to control glomerular isolates was found.Thus, the mesangium in normal animals functions in a manner analogous to that of the general reticuloendothelial system. In nephrotic rats the mesangial uptake of macromolecules is makedly increased, a finding not observed in other tissues.     

Specific binding of soluble fibrin to macrophages

Guinea pig peritoneal macrophages were demonstrated to bind selectively soluble 125I-fibrin and fibrin/fibrinogen complexes as compared with fibrinogen, fibrinogen degradation products, and fibrin degradation products. Cellular uptake was considered to be surface receptor binding on the basis of removal of bound 125I-fibrin by trypsin and because uptake occurred in the presence of metabolic inhibitors. 125I-fibrin uptake could be blocked by nonradioactive fibrin but not by IgG or immune complexes. Binding was uneffected by prior treatment with plasmin or trypsin but was calcium dependent. Only limited reversibility of binding could be demonstrated after prolonged incubation. Scatchard plots permitted an estimate of the number of bound molecules. At saturation 6.92 X 10(6) 125I-fibrin molecules were bound per cell. Similar binding of fibrin was noted in polymorphonuclear leukocytes, but not lymphocytes or fibroblasts. Soluble fibrin binding may be a host defense mechanism whereby the reticuloendothelial system can remove fibrin from the blood before the development of microthrombi.     

Experimental Membranous Glomerulonephritis in Rats: QUANTITATIVE STUDIES OF GLOMERULAR IMMUNE DEPOSIT FORMATION IN ISOLATED GLOMERULI AND WHOLE ANIMALS

Quantitation of immune deposit formation in glomeruli and correlation with immunohistologic and functional changes has been accomplished only in models of anti-glomerular basement membrane antibody-induced nephritis, or indirectly in immune complex disease by measuring radiolabeled antigen deposition. The kinetics of subepithelial immune deposit formation and the relationship between the quantity of antibody deposited and proteinuria are defined here for the first time in an established model of membranous immune complex nephritis (passive Heymann nephritis) induced by a single intravenous injection of ¹²⁵I-labeled sheep immunoglobulin (Ig)G antibody to rat tubular brush border antigen (Fx1A). Measurement of antibody deposition in glomeruli (GAb) isolated from rats injected with 10 mg of anti-Fx1A demonstrated a mean of 12 μg GAb in 4 h, which increased linearly to 48 μg in 5 d. GAb represented only 20 and 44% of total kidney antibody binding at these times. Proteinuria occurred only after 4-5 d of antibody deposition in rats with total kidney antibody binding exceeding ~200 μg/2 kidneys. Steroid treatment and vasoactive amine blockade did not significantly alter the quantity or localization of immune deposits. It was also demonstrated that isolated rat glomeruli specifically bound nephritogenic quantities of anti-Fx1A in vitro within hours. Analysis of the quantitative aspects of glomerular antibody deposition in vivo and glomerular antibody binding in vitro provides additional evidence that subepithelial immune deposits in passive Heymann nephritis may form in situ by reaction of free antibody with antigenic constitutents of the normal rat glomerulus. The observed kinetics of deposit formation differ markedly from those in anti-glomerular basement membrane disease and suggest a role for factors in addition to antigen-antibody interaction in determining this unique pattern of glomerular immune deposit formation.     

Intrarenal platelet consumption in the diffuse proliferative nephritis of systemic lupus erythematosus.

1.Platelet survival and an index of the localization of platelets in the kidney were studied in patients with the proliferative nephritis of systemic lupus erythematosus, either focal or diffuse, and in control subjects. Platelet survival was reduced in patients with proliferative lupus nephritis, more in those with diffuse rather than focal renal involvement. 2. The index of renal platelet localization in patients with diffuse proliferative nephritis suggested an intrarenal platelet consumption not found in other groups. 3. A patient with the classical platelet autoantibody disease, idiopathic thrombocytopenic purpura, also showed reduced platelet survival but localization of platelets was in the spleen rather than the kidney. 4. Intrarenal platelet consumption in diffuse proliferative lupus nephritis may be an epiphenomenon of pre-existing scarring or platelet aggregation secondary to immune complex-formation, which contributes to the progressive sclerosing lesions of this form of nephritis.     

Single and multiple drug therapy in autologous immune complex nephritis in rats.

Autologous immune complex (AIC) nephritis is a form of chronic renal disease with remarkable similarities to idiopathic membranous nephropathy occurring in man. AIC nephritis was induced in 160 gram Lewis rats with a single footpad injection of tubular brush-border antigen (FxIA) in complete Freund's adjuvant. When killed at 8 weeks, 85 per cent of the rats demonstrated typical diffuse glomerular deposits of immunoglobulin G and B1C (C1/3 component of complement) by immunofluorescent microscopy, and subepithelial electron-dense deposits by electron microscopy. Both immune complex disease and significant proteinuria occurred in two-thirds of these animals. An attempt to modify the natural course of established AIC nephritis using large doses of potent glucocorticoids (methyl-prednisolone), anti-inflammatory agents (acetylsalicylic acid, indomethacin, and cyproheptadine), and immunosuppressive drugs (cyclophosphamide, azathioprine) was begun 4 weeks after initial immunization and continued for 4 more weeks. None of the single drug nor multiple drug protocols employed was of demonstrable benefit in ameliorating the immune events operating in AIC nephritis. Cyclophosphamide and indomethacin, when used singly, were associated with significant mortality in the animals studied. All combined drug protocols involving glucocorticoids and antimetabolites were associated with unacceptable mortality as well. Of interest, immune complexes could not be demonstrated in the vascular choroid plexus of any rat with AIC nephritis. This failure to modify the course of established renal disease (AIC) in an experimental animal with generally available pharmacologic agents, is similar to the usual results of such treatment in chronic renal disease (idiopathic membranous nephropathy) in man. It is possible that new and more potent anti-inflammatory agents employed singly or in various combinations, will permit more successful manipulation of the host's immunologic system to prevent or modify immune injury of the renal glomerulus.     

Secondary nephrotic syndrome due to collagen disease and vasculitis

The most frequent and representative nephrotic syndrome associated with collagen disease is encountered in patients suffering from lupus nephritis. Lupus nephritis is a glomerulonephritis, which discloses various localizations of immune complexes in the endothelium, mesangium and subepithelium. In addition, vasculitides complicated by nephrotic syndrome also show the deposition of immune complexes in their glomeruli, such as Henoch-Sch?nlein nephritis and cryoglobulinemic nephritis. The pathogenetic mechanisms of these nephrotic syndromes are explained as follows. The depositions of immune complexes in glomeruli causes proteinuria through a variety of mechanisms. Namely, subendothelial and mesangial immune deposits give capillary and mesangial injuries as well as inflammation that are mediated through activation of complements and cytokines, and subsequently leads to nephrotic-range proteinuria and impairment of renal function. On the other hand, subepithelial and intramembranous deposits disrupt the regulated arrangement of epithelial cells and slit diaphragms, and then disturb the slit diaphragms. The eventual dysfunction of slit diaphragms accordingly progresses to massive proteinuria even without capillary injury. Therefore, nephrotic syndrome associated with collagen disease or vasculitis is usually observed in lupus nephritis or vasculitis related to immune complex depositions, but is not observed in non-immune complex glomerulopathy or vasculopathy.     

Induction of Membranous Nephropathy in Rabbits by Administration of an Exogenous Cationic Antigen: DEMONSTRATION OF A PATHOGENIC ROLE FOR ELECTRICAL CHARGE

We examined the role of antigenic electrical charge as a determinant of glomerular immune complex localization in the rabbit. Serum sickness nephritis was induced in groups of New Zealand white rabbits by daily 25-mg intravenous injections of bovine serum albumin (BSA) chemically modified to be cationic (pI > 9.5) or more anionic (pI, 3.5-4.6); an additional group received unmodified native BSA (pI, 4.5-5.1). Factors known to influence immune complex localization, e.g., molecular size of the administered antigen and resulting circulating immune complexes, immunogenicity, and disappearance time from the circulation were examined and found to be similar for both anionic and cationic BSA. Charge modification did increase the nonimmune clearance of cationic and anionic BSA compared with native BSA. Injected cationic BSA was shown in paired label experiments to bind directly to glomeruli compared with native BSA. The renal lesion produced by cationic BSA was markedly different from that found in rabbits given anionic or native BSA. Animals receiving cationic BSA uniformly developed generalized diffuse granular capillary wall deposits of IgG, C3, and BSA detected after 2 wk of injections and increasing until death at 6 wk. Qualitatively similar deposits were produced by the administration of low doses of cationic BSA of only 1 or 10 mg/d. In contrast, the injection of both anionic and native BSA resulted in mesangial deposits at 2 and 4 wk with capillary wall deposits appearing by 6 wk. Ultrastructural examination of animals receiving cationic BSA revealed pure, extensive formation of dense deposits along the lamina rara externa of the glomerular basement membrane whereas such deposits were absent or rare in animals injected with the anionic or native BSA. Albuminuria was significantly greater at 6 wk in the groups receiving cationic BSA with a mean of 280 mg/24 h compared with 53 mg/24 h in the combined groups injected with anionic or native BSA. Blood urea nitrogen values were similar in all groups at 2 and 4 wk but higher in the animals receiving cationic BSA at 6 wk.     

STUDIES IN MOLECULAR PATHOLOGY : I. LOCALIZATION AND PATHOGENIC ROLE OF HETEROLOGOUS IMMUNE COMPLEXES

After intravenous injection in mice, rabbit immune complexes, solubilized in antigen excess and containing fluorescent antigens (BSA* or OA*) or fluorescent antibody, or both, were promptly localized in reticuloendothelial cells, and polymorphonuclear leukocytes, of the sinusoids of liver and the red pulp of spleen; in glomeruli and elsewhere in kidney; in capillary endothelium of heart and lung; and in hepatic cells. Thereafter manifold processes occurred. Within 48 hours the immune complexes were scarcely detectable in liver and splenic red pulp but now were localized in the germinal centers of white pulp where heretofore they had been seen only in trace amounts. This new localization presumably was associated with the antibody-forming activity of the germinal centers, for the immune phase of antigen clearance from the blood had already begun. Although the immune complexes were localized in various regions of the nephrons and their appertaining blood vessels, the initial sites of predilection were the glomerular capillary walls and intercapillary spaces. After 48 hours the immune complexes were still detectable, although in diminished amounts, in the glomeruli but had by now essentially disappeared from other renal sites. The localization of immune complexes in the kidney was associated with proteinuria and with structural changes which closely simulated in some instances those of human membranous glomerulonephritis, of focal and diffuse types, and consisted mainly of eosinophilic swellings of the glomerular capillary walls, intercapillary spaces, and basement membranes. There was a close correspondence between the distributions of the eosinophilic swellings and the fluorescent immune complexes. The renal localization and persistence of fluorescent antigens (BSA* or OA*), after separate injections in mice, differed from that of fluorescent immune complexes in several respects. For example BSA* showed predilection for the glomerular basement membranes and was localized sparsely in the capillary walls and intercapillary spaces; OA* was localized only in minute amounts; and neither was detectable in more than trace amounts at 48 hours after injection. These fluorescent proteins (of low molecular weights, 40,000, 70,000) did not cause glomerulonephritis within the time interval studied, whereas fluorescent immune complexes, containing on the average two molecules of antigen to one of antibody (with minimum molecular weights of 240,000 to 300,000) produced glomerulonephritis in some instances, in confirmation of the observations of others. Since the localization of the immune complexes occurred immediately and without known immunologic relation to the kidney itself, the selective physical retention of proteins by structures comprising the glomerular ultrafilters appeared to be of pathogenic significance in this form of membranous glomerulonephritis in mice, as perhaps also in nephrotic glomerulonephritis in man. If after injection of fluorescent immune complex, homologous antiserum was also administered intravenously so as to produce acute anaphylactic death, coarse and occlusive depositions of immune precipitates occurred in pulmonary, myocardial, and renal capillaries, and in hepatic sinusoids.     

Accelerated immune complex nephritis due to mesangial overloading in spontaneous hypertensive (SHR) rats.

Immune complex nephritis induced by bovine serum albumin (BSA) and horseradish peroxidase (HRP) was superimposed upon spontaneous hypertensive rats (SHR rats) which were pretreated with polyvinyl alcohol (PVA). PVA accumulated predominantly in the mesangium, and the superimposed nephritis developed more accelerated glomerular damage with marked capillary deposition of immune complexes than control animals which were not pretreated with PVA.     

In vivo degradation of immune complexes in the kidney by orally administered enzymes

Preformed immune complexes were deposited in kidney glomeruli of rabbits after i.v. injection. In vivo disintegration of these complexes by orally administered enzymes was investigated. 3 rabbits were given labelled trypsin and papain and the radioactivity and enzyme activity determined in the blood. The radioactive fraction showed an active enzyme concentration of 3-5 mg%. 13 experimental rabbits and 3 control animals received three i.v. injections of 5 ml preformed soluble immune complexes at 12-hour intervals. 24 hours after the last injection the experimental animals were fed 1600 mg enzyme mixture. All animals were sacrificed 4 hours later and the glomeruli of the kidney were investigated by immunofluorescence. All control animals showed large amounts of immune complexes in the glomeruli. Experimental animals, which had all received oral enzymes showed no immune complexes any more, or only residual immune complexes in some glomeruli. This observation pointed to in vivo disintegration of immune complexes by orally-administered enzymes as providing the basis for the treatment of immune complex diseases.     

Low density lipoprotein metabolism by human macrophages activated with low density lipoprotein immune complexes. A possible mechanism of foam cell formation

Human macrophages play a key role in atherogenesis and are believed to be the progenitors of the cholesteryl ester (CE)-laden foam cells present in early atherosclerotic lesions. Several mechanisms by which macrophages accumulate CE have been recently described. One involves a perturbation in LDL metabolism subsequent to macrophage activation. Thus, we decided to study the effect of macrophage activation by immune complexes on N-LDL metabolism. Initially, LDL-containing immune complexes (LDL-IC) were chosen, since increased plasma levels of these IC have been reported in patients with coronary heart disease. Human macrophages stimulated for 22 h with LDL-IC (250 micrograms/ml) and incubated afterwards for 20 h with 10 micrograms/ml 125I-N-LDL showed a six- and fourfold increase in the accumulation and degradation, respectively, of 125I-N-LDL over the values observed in nonstimulated cells. Scatchard analysis of 125I-N-LDL-specific binding suggests an increase (20-fold) in the number of LDL receptors in macrophages stimulated with LDL-IC. We studied other immune complexes varying in size and antigen composition. Some of the IC were able to stimulate, although to a lesser degree, the uptake of N-LDL by macrophages. Lipoprotein IC are more efficient and have the greatest capacity to increase N-LDL uptake and CE accumulation. We conclude that human macrophage activation by LDL-IC leads to an increase in LDL receptor activity and promotes in vitro foam cell formation.     

Intravascular blood coagulation and immune complex inflammation in patients with systemic lupus erythematosus

Immune complex inflammation associated with systemic lupus erythematosus (SLE) is attended by activation of the coagulation system up to the development of disseminated intravascular blood coagulation (the DIC syndrome). Study of the hemocoagulation in 106 patients with SLE revealed the signs of the chronic DIC syndrome that manifested itself largely by hypercoagulation, increased thrombin formation and proneness to inhibition or activation of fibrinolysis. These alterations were more demonstrable in patients with a highly active condition and lupus nephritis. The clinical symptoms of the chronic DIC syndrome in the form of hemorrhages and thromboses were seen in 27 patients. Correlations were discovered between the level of soluble fibrin-monomer complexes and characteristics of inflammatory and immunologic activity. Therefore, a close interrelationship has been shown between immune complex processes and alterations in the hemocoagulation associated with SLE. It has been also demonstrated that the DIC syndrome plays a role in the progression of lupus nephritis.     

Circulating immune complexes detected by 125I-Clq deviation test in sera of cancer patients.

The presence of circulating immune complexes in freshly drawn sera of patients with various forms of malignancies was detected by the 125I-Clq deviation test of Sobel et al. More than 50% of the 459 cancer sera showed a high inhibition of 125I-Clq uptake by sensitized sheep erythrocytes when compared with sera of 50 healthy laboratory personnel. The levels were compared with levels of total hemolytic complement and immunochemical determinations of Cl1 and C3. A correlation between high levels of circulating immune complexes and low levels of Clq was suggested. These immune complexes were separated by sucrose density gradient ultracentrifugation at low pH and were found to be heavier than 19S. Fluctuation of levels of immune complexes was evident when serial samples from the same patient were tested. Decrease of levels of immune complexes and a concomitant increase of Clq were detected after Calmette-Gueérin bacillus and autologous tumor cell treatment in some melanoma patients.     

Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq

A sensitive and reproducible procedure for the detection of soluble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled C1q is reacted with sera containing immune complexes. Separation of free from complex bound [(125)I]C1q is achieved by selective precipitation with polyethylene glycol (PEG). The method is based on both the large molecular size and the C1q-binding property characterizing immune complexes. The minimal amount of aggregated immunoglobulins thus detected is about 10 mug and that of soluble human IgG-anti-IgG complexes is about 3 mug of complexed antibody. Some immune complexes formed in large antigen excess (Ag(2)Ab) can still be detected by this radiolabeled C1q binding assay. The specificity of the radiolabeled C1q binding test was documented by the inability of antigen-F(ab')(2) antibody complexes to lead to a precipitation of [(125)I]C1q in PEG.In a second step, this radiolabeled C1q binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes.Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the hepatitis B antigen (HB-Ag). Significantly increased [(125)I]-C1q binding values were observed in 52 sera from SLE patients when compared to values obtained with healthy blood donors (P<0.001). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients.No increased [(125)I]C1q binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [(125)I]C1q percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [(125)I]C1q. The results were also used for a correlative study of [(125)I]C1q binding to IgG levels in the sera but increased [(125)I]C1q binding could not be attributed to high serum IgG levels which are likely to account for gammaglobulin aggregates.These examples suggest the utility of the radiolabeled C1q binding assay for the evaluation of immune complex diseases in human pathology.     

Requirement and role of C5a in acute lung inflammatory injury in rats.

The complement activation product, C5a, may play a key role in the acute inflammatory response. Polyclonal antibody to rat C5a was used to define the requirements for C5a in neutrophil-dependent inflammatory lung injury after systemic activation of complement by cobra venom factor (CVF) or after intrapulmonary deposition of IgG immune complexes. In the CVF model, intravenous infusion (but not intratracheal instillation) of anti-C5a produced a dose-dependent reduction in lung permeability and in lung content of myeloperoxidase. In C6-deficient rats, CVF infusion caused the same level of lung injury (measured by leak of 125I-albumin) as found in C6-sufficient rats. In the IgG immune complex model of lung injury, anti-C5a administered intratracheally (but not intravenously) reduced in a dose-dependent manner both the increase in lung vascular permeability as well as the buildup of lung myeloperoxidase. Treatment with anti-C5a greatly suppressed upregulation of lung vascular intercellular adhesion molecule-1 (ICAM-1). This was correlated with a substantial drop in levels of TNFalpha in bronchoalveolar fluids. These data demonstrate the requirement for C5a in the two models of injury. In the IgG immune complex model, C5a is required for the full production of TNFalpha and the corresponding upregulation of lung vascular ICAM-1.     

Macrophage activation: increased ingestion of IgG-coated erythrocytes after administration of interferon inducers to mice

In vitro phagocytosis of IgG-opsonized sheep erythrocytes (EA) was used to measure the in vivo activation of mouse peritoneal macrophages. Uptake of EA as enhanced by the extraperitoneal administration of Newcastle disease virus, vesicular stomatitis virus, tilorone or polyinosinic-polycytidylic acid. Ingestion of EA was similarly stimulated by lipopolysaccharide or killed Corynebacterium parvum. Dose- response curves relating concentrations of IgG to phagocytosis were parallel for both treated and control animals. This indicates that the heterogeneity of the macrophage populations did not change and that the overall populations were activated with respect to phagocytic ability. Numbers of macrophages were not increased (except in C. parvum-treated mice), suggesting that resident, rather than newly recruited macrophages, were activated by the different agents.     

Hepatic and intestinal blood flow following thermal injury

Because cardiac output decreases after burn injuries, investigators have assumed, based upon dye clearance techniques, that hepatic and intestinal blood flow are also decreased following these injuries. Blood flow to the liver, stomach, small intestine, and kidney was determined by the uptake of 201thallium and 125I-labeled fatty acid (para-125I-phenyl-3-methyl pentanoic acid) in a 20% body surface area scald injury that also included plasma volume replacement resuscitation. Uptake of these radioisotopes was determined 15 minutes, 18 hours, and 72 hours after injury. The uptake of the 201thallium and 125I-labeled fatty acid by the gastrointestinal tissues was not statistically different at any of the time periods after comparison of the injured and control (sham-treated) animals. 201Thallium uptake by the kidney was significantly diminished 15 minutes after the burn injury (P less than 0.01). Based on these blood flow measurement techniques, the data suggest that the 20% body surface area scald injury did not alter blood flow to the liver or gastrointestinal tract within the initial 72 hours after the burn injury even though a decrease in renal blood flow was easily detected. These results suggest that the dysfunction of the gastrointestinal system or hepatic system observed after an acute burn injury is not simply the result of hypovolemic shock, which reduces both renal and mesenteric blood flow. These gastrointestinal and hepatic alterations may be related to a factor or factors other than intestinal ischemia.     

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.     

Influence of antigen on immune complex behavior in mice.

To explore the possibility that the behavior of immune complexes can, under some circumstances, be directed by the antigen, we have studied the behavior of complexes of identical size made with the glycoproteins, orosomucoid (OR), and ceruloplasmin: or with their desialylated derivatives, asialo-orosomucoid (ASOR) and asialo-ceruloplasmin. Such desialylated proteins are rapidly removed from the circulation by a hepatic cell receptor for galactose, the sugar exposed upon removal of sialic acid. Mixtures of 125I-goat anti-ASOR with either ASOR or OR and mixtures of 125I-rabbit anti-OR with either ASOR or OR form complexes identically. The complexes were separated by density gradient centrifugation and injected intravenously into C3H mice. Blood clearance and hepatic uptake of the OR complexes and ASOR complexes were markedly different. T 1/2 for the goat OR complexes exceeded 300 min, whereas that for the ASOR complexes was 15 min. More detailed studies using rabbit complexes of various sizes revealed that light rabbit complexes behaved similarly to the goat complexes. The light rabbit OR complexes were cleared slowly, with only 18% found in the liver at 60 min, whereas the light rabbit ASOR complexes were cleared much more rapidly, with 62% found within the liver by 30 min. This rapid clearance was completely suppressed by a prior injection of a blocking dose of ASOR, which implies uptake by a galactose-mediated mechanism on hepatocytes. As the size of the rabbit complexes increased, so did the rate of Fc receptor-mediated clearance. Heavy rabbit OR complexes were cleared more rapidly than light OR complexes but not so rapidly as heavy ASOR complexes. The clearance and hepatic uptake of the heavy OR complexes were markedly suppressed by a prior injection of heat-aggregated gamma globulin, a known Fc receptor-blocking agent (45% hepatic uptake without and 6% with aggregated gamma globulin). The heavy rabbit ASOR complexes exhibited inhibition of blood clearance and hepatic uptake by both galactose receptor-blocking and Fc receptor-blocking agents. A blocking dose of ASOR reduced the hepatic uptake at 30 min from 75 to 49%, and heat-aggregated gamma globulin reduced it from 75 to 39%, which suggests that these heavy complexes were removed from the circulation by receptors both for the immunoglobulin and for the antigen. Cell separation studies and autoradiographs confirmed that those complexes cleared primarily by galactose-mediated mechanism were within hepatocytes, and those cleared by Fc receptors were within the nonparenchymal cells of the liver. It seems probable, therefore, the some antigen-antibody complexes may be removed from the circulation via receptors not only for immunoglobulin but also for antigen.