Circulating immune complexes in the serum of diabetes mellitus in childhood by a modified 125I-C1q binding test.

The 125I-C1q binding test for the detection of soluble immune complexes in native unheated human serum was applied to the study of sera from 52 patients with diabetes mellitus in childhood. This radiolabeled C1q binding test is more sensitive and reproducible among the various methods proposed for the detection of immune complexes. The 125I-C1q binding activity in 52 sera from diabetes mellitus in childhood was 9.47 +/- 0.36% compared to 6.94 +/- 0.74% in normal controls. 125I-C1q binding values in diabetes mellitus in childhood were significantly higher than normal controls. Slight high values were seen in 3 patients with positive anti-DNA-antibodies in diabetes mellitus in childhood. 125I-C1q binding was not significantly increased in patients with positive antithyroid antibodies and insulin antibodies. There was no significant correlation between the duration of diabetes and 125I-C1q binding activity.     

Circulating immune complexes in Graves' disease.

Circulating immune complexes in Graves' disease sera were detected by the 125I Clq deviation test. High titers of immune complexes were detected and correlated significantly with the microsomal antibody but not with the thyroglobulin antibody titer nor with serum thyroxine levels. Serum fractionation studies in a patient with high titer of immune complexes revealed these to be heterogeneous in size, sedimenting in 19S, intermediate and 7S regions. The data suggest a role for immune complexes in the pathogenesis of Graves' disease.     

Circulating immune complexes in patients with breast cancer.

In a retrospective study in women with breast cancer circulating immune complex levels were measured by radioimmunoprecipitation with 125I-Clq. Before operation all the patients showed plasma immune complex levels significantly higher than those in controls. Twelve months after mastectomy patients identified clinicopathologically as having a good prognosis had almost normal levels of immune complexes. By contrast, patients with detectable dissemination on diagnosis or those who died within 22 months after mastectomy had significantly raised plasma levels. The tumour-specific nature of the immune complexes detected remains to be shown and suggestions about the applicability of this test not only for prognosis but also for monitoring the course of malignant diseases need to be confirmed by further investigations.     

Only the initial binding of cationic immune complexes to glomerular anionic sites is mediated by charge-charge interactions

The role of charge-charge interactions between cationic immune complexes and the anionic sites on the glomerular basement membrane was examined. For this purpose, soluble immune complexes at fivefold antigen excess were prepared with human serum albumin and cationized rabbit antibodies to this protein. When unrelated cationic proteins, protamine sulfate or cationized rabbit serum albumin, were given 1 min before the cationized immune complexes, glomerular immune deposits did not form. Cationic immune complexes allowed to deposit in glomeruli could readily be displaced by protamine sulfate or cationized rabbit serum albumin injected 1 min after the immune complexes. If the same cationic molecules were injected 1 hr after the immune complexes, the complexes could not be displaced from glomeruli. In contrast, cationic complexes that were deposited in glomeruli in the presence of a very high degree of antigen excess in circulation to prevent their condensation into larger complexes in glomeruli were readily displaced at 1 min and 1 hr with protamine sulfate or with cationized rabbit serum albumin. On the basis of these results, we concluded that the initial binding of cationic immune complexes to glomeruli occurs by charge-charge interactions. Once the immune complexes in glomeruli condense to larger deposits, forces other than charge-charge interactions are responsible for their retention in glomeruli.     

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.     

Divalent cations regulate glucagon binding. Evidence for actions on receptor-Ns complexes and on receptors uncoupled from Ns

The effects of Mg2+ or ethylenediaminetetraacetic acid (EDTA) on 125I-glucagon binding to rat liver plasma membranes have been characterized. In the absence of guanosine 5'-triphosphate (GTP), maximal binding of 125I-glucagon occurs in the absence of added Mg2+. Addition of EDTA or Mg2+ diminishes binding in a dose-dependent manner. In the presence of GTP, maximal binding occurs in the presence of 2.5 mM Mg2+ (EC50 = 0.3 mM) while EDTA or higher concentrations of Mg2+ diminish binding. Response to exogenous Mg2+ or EDTA depends on the concentration of Mg2+ in the membranes and may vary with the method used for membrane isolation. Solubilized 125I-glucagon-receptor complexes fractionate on gel filtration columns as high molecular weight, GTP-sensitive complexes in which receptors are coupled to regulatory proteins and lower molecular weight, GTP-insensitive complexes in which receptors are not coupled to other components of the adenylyl cyclase system. In the absence of GTP, 40 mM Mg2+ or 5 mM EDTA diminishes receptor affinity for hormone (from KD = 1.2 +/- 0.1 nM to KD = 2.6 +/- 0.3 nM) and the fraction of 125I-glucagon in high molecular weight receptor-Ns complexes without affecting site number (Bmax = 1.8 +/- 0.1 pmol/mg of protein). Thus, while GTP promotes disaggregation of receptor-Ns complexes, Mg2+ or EDTA diminishes the affinity with which these species bind hormone. In the presence of GTP, hormone binds to lower affinity (KD = 9.0 +/- 3.0 nM), low molecular weight receptors uncoupled from Ns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes

We have studied the Fc receptor-mediated pinocytosis of immunoglobulin G (IgG)-containing immune complexes by mouse macrophages. IgG complexes were formed from affinity-purified rabbit dinitrophenyl IgG and dinitrophenyl modified BSA at molar ratios of 2.5-10:1. Both the specificity of binding and the fate of internalized receptors were analyzed using monoclonal and polyclonal anti-Fc receptor antibodies. Based on the susceptibility of surface-bound ligand to release by proteolysis, we have found that at 37 degrees C, 125I-labeled IgG complexes were rapidly internalized (t1/2 less than 2 min) and delivered to lysosomes; acid-soluble 125I was detectable in the growth medium within 5-10 min of uptake. However, kinetic evidence indicated that Fc receptors were not efficiently re-used for multiple rounds of ligand uptake. Instead, macrophages that were exposed continuously to saturating concentrations of IgG complexes exhibited a selective and largely irreversible removal of Fc receptors from the plasma membrane. This loss of surface receptors correlated with an increased rate of receptor turnover, determined by immune precipitation of Fc receptors from 125I-labeled macrophages. Thus, in contrast to the results obtained in the accompanying paper (I. Mellman, H. Plutner, and P. Ukkonen, 1984, J. Cell Biol. 98:1163-1169) using a monovalent ligand, these data indicate that the interaction of Fc receptors with polyvalent complexes leads to the degradation of both ligand and receptor following their delivery to lysosomes.     

In vivo and in vitro studies of the binding of antibody/dsDNA immune complexes to rabbit and guinea pig platelets

The in vivo and in vitro binding of prepared antibody/dsDNA immune complexes to rabbit and guinea pig cellular blood components was examined. The in vitro binding in these two nonprimates was almost entirely due to platelets, and required homologous, intact complement; furthermore, no appreciable binding was observed for neutrophils, mononuclear cells, or erythrocytes at normal blood concentrations. The in vivo binding reaction occurred quite rapidly (less than 1 min for maximal binding) and the majority of the injected counts were cleared from the circulation in 3 to 5 min. Over this time period, however, a large fraction of the counts remaining in the circulation also remained bound to the animals' cells (presumably platelets), and this result was most pronounced for complement-fixing immune complexes prepared with high m.w. dsDNA. In vitro studies confirmed that immune complexes prepared with such dsDNA are rather slowly released from the animal platelets in the presence of homologous serum, and this result is in marked contrast to the considerably greater lability of bovine serum albumin/anti-bovine serum albumin immune complexes that are bound to complement receptors on animal and human cells. These observations suggest that the fate of immune-complexed dsDNA in the circulation may be very different from that of free dsDNA, and in the case of nonprimates may involve a platelet-mediated immune complex clearance mechanism analogous to the erythrocyte-mediated immune complex clearance mechanism which is believed to be operative in primates.     

The complement-mediated binding of soluble antibody/dsDNA immune complexes to human neutrophils

The complement-mediated binding of soluble antibody/3H-dsDNA immune complexes (prepared in vitro) to human polymorphonuclear leukocytes (PMN) has been investigated quantitatively. Studies with isolated complement components in conjunction with experiments on the binding of these complexes to human red blood cells suggest that the binding to both cell types is mediated predominantly by CR1 (C4b-C3b) receptors but that CR3 (iC3b or C3d-g) receptors may play a role in binding to PMN but probably not to RBC. Our results also indicate that under the standard conditions of these assays (37 degrees C, 20 to 40 min incubations) there is no significant internalization of the soluble antibody/dsDNA immune complexes after they are bound by the PMN.     

Mechanism of binding of soluble immune complexes to lymphocytes

Soluble immune complexes prepared with either (a) ¹²⁵I BSA and mouse antiserum to BSA in the presence of fresh mouse serum (AgAbC) or with (b) ¹²⁵I BSA and a 7S fraction of the mouse antiserum to BSA (AgAb) bind to a certain proportion of mouse lymph node and spleen lymphocytes (but not to thymocytes). The efficiency of binding is greater when complexes are prepared at defined antigen/antibody ratios and when the incubation with lymphocytes is performed at 37 °C. However, a significant degree of binding occurs at lower temperatures and even at 0 °C. Cells which bind soluble complexes overlap extensively with complement receptor lymphocytes (CRL) (B lymphocytes) since the specific elimination of CRL also depletes the population of cells which can bind soluble complexes. Two types of interactions are involved in the binding: one mediated by antibody which has been aggregated by antigen and another by complement (probably C3). They can be operationally distinguished because (1) after treatment of the lymphocytes with trypsin, the binding of AgAbC (but not of AgAb) is strongly inhibited; (2) the binding of AgAb (but not of AgAbC) is inhibited by heat-aggregated Ig; (3) the binding of AgAbC (but not of AgAb) to lymphocytes inhibits their subsequent interaction and rosette formation with erythrocytes sensitized by antibody and complement components; and (4) cobra venom factor markedly alters the binding of AgAbC to lymphocytes.     

The determination of antigen-specific immune complexes by an immunoenzyme method]

The methodological approach permitting the detection of immune complexes containing specific antibodies to a definite antigen in the enzyme-linked immunosorbent assay (ELISA) is described. The basic conditions of the assay were optimized. Immune complexes were precipitated from blood serum with 3.5% polyethylene glycol 6000 for 4 hours. The precipitate thus obtained was dissolved and incubated in polystyrene plates with immobilized antigen at 37 degrees C for a long time (at least 6 hours) in a humid chamber. The amount of bound antibodies, determined by ELISA techniques, was conjectured from the level of antigen-specific immune complexes. The proposed approach can be used in the immunodiagnosis of infectious diseases.     

A receptor for formaldehyde-treated serum albumin on human placental brush-border membrane

Formaldehyde-treated serum albumin (f-Alb) is known to be taken up and degraded by sinusoidal liver cells via receptor-mediated endocytosis. We report that 125I-labeled f-Alb (125I-f-Alb) binding to human placental brush-border membranes also occurs. This binding reached equilibrium within 40 min at 37 degrees C. Kinetic studies demonstrated the presence of saturable binding with an apparent Kd of 2.1 micrograms of f-Alb/ml and a maximal binding of 2.3 micrograms/mg of membrane protein at pH 7.5. Maximal binding was observed at between pH 7.5 and 8.0. 125I-f-Alb binding to the membranes was little inhibited by a 1000-fold molar excess of ovalbumin, human apo-transferrin and native bovine serum albumin. No binding was observed with membranes which had been pretreated with proteinase or trypsin. This f-Alb receptor was extremely heat-stable, since the binding was not abolished even by pretreatment of the membranes at 78 degrees C for 30 min. EDTA, Ca2+ and Mg/4 had no effect on 125I-f-Alb binding, so the binding was independent of divalent cations. These data suggest that a receptor specific for f-Alb exists on human placental brush-border membranes of syncytial trophoblasts.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

Circulating immune complexes in cervical cancer patients as detected by C1q binding

In a retrospective study in women with cervical cancer, circulating immune complex levels were measured by radioimmunoprecipitation with 125I-C1q. Sera from 46 patients with cervical cancer and 35 normal controls were examined. Significantly higher levels of immune complexes were detected in cancer patients compared with controls. Mean value of binding capacity in patients was 49.8%, and by contrast, in the controls was 27.4% (two-tail test = 0). Increases in tumor mass were associated with high levels of circulating immune complexes. The presence of immune complexes in circulation statistically correlated with disease activity, however, the assay used still had limited value for diagnosis or aiding in therapeutic decisions. Nevertheless, the future holds promise for such uses.     

Role of immune complexes in the humoral leukocyte adherence inhibition test

Summary The humoral leukocyte adherence inhibition (H-LAI) assay has recently been found to measure an antitumor immunefactor. In this assay, trypsinized leukocytes from control persons are used as indicator cells and 0,25% serum from the patient is added to the assay system together with the relevant tumor antigen.In the present work, evidence is presented that the H-LAI response is mediated through in vitro-formed immune complexes. Different antibody-antigen pairs (anti-albumin — albumin; anti-₂microglobulin — ₂microglobulin; anti-carcinoembryonic antigen — carcinoembryonic antigen; anti-transferrin — transferrin) have been added to the assay mixture. A significant H-LAI response was observed when immune complexes were formed. On the other hand, when unrelated antibody — antigen pairs were added, no response was found. The specificity was demonstrated in experiments where two different antibodies were added simultaneously and the response tested both against the two corresponding antigens and against unrelated antigens.Since the same trypsinized indicator cells can be used for different immune complexes, it is likely that the response is mediated through common receptors on the cell surface with affinity for immune complexes, i.e., Fc-receptors. Presumably, the H-LAI test gives response to immune complexes in general and is as such not specific. The specificity is achieved through the addition of specific antigen and the subsequent in vitro formation of immune complexes.     

Immunohistochemical detection of Fc receptor. II. Electron microscopic demonstration of Fc receptor by using soluble immune complexes of ferritin-antiferritin immunoglobulin G.

The mouse mesenteric lymph node cells were incubated in the soluble immune complexes of ferritin-antiferritin immunoglobulin G at 37 degrees C for 20 min. After being washed, postfixed with OsO4 and dehydrated by degraded ethanol series, the lymph node cells were observed by electron microscope. Apprroximately 15% of the cells (mainly composed of small lymphocytes) bound ferritin particles to the cell surface. The distribution pattern of the binding of ferritin particles (ferritin-antiferritin immunoglobulin G) took the form of discrete patches of irregular distribution interspaced with unlabeled portions. The electron microscopic features of ferritin particles (ferritin-antiferritin immunoglobulin G) attached to the cell surface suggest that a structure of constant conformation (Fc receptor) situated in the cell membrane takes part in the binding of ferritin-antiferritin immunoglobulin G.     

Isolation and characterization of circulating immune complexes from rats with experimental membranous nephropathy

Circulating immune complexes (CIC) were isolated from serum from controls and rats with active Heymann nephritis (n = 31) by two methods. CIC detected by the fluid phase Clq binding assay were precipitated from serum using Clq and polyethylene glycol. CIC were also isolated by sequential chromatography with anion exchange and lectin affinity supports. The isolated material was analyzed by PAGE and immunoblotting. The immune complex material isolated by both methods from rats with Heymann nephritis contained the same 60/65-kDa tubular Ag. By immunoblotting, the 60/65-kDa tubular Ag-bound antibodies from rats with active Heymann nephritis, but not antibodies to gp330. Antibody bound to the 60/65-kDa tubular protein in the CIC was isolated. This antibody bound to a similar Ag in glomerular eluates from rats with active Heymann nephritis when tested by immunoblotting. These observations suggest that glomerular immune deposits and CIC in rats with Heymann nephritis contain the same tubular Ag. The 60/65-kDa Ag was isolated from CIC by HPLC using anion exchange and hydrophobic interaction columns. Rats immunized with this Ag developed Heymann nephritis. These studies suggest that CIC contribute to the development of glomerular subepithelial immune deposits in this model of membranous nephropathy. These studies do not exclude the participation of other Ag-antibody systems in Heymann nephritis, including gp330. This report describes methods for isolation and characterization of Ag-antibody components of CIC that might be useful to studies of other immune complex-mediated diseases.     

ATP-dependence of 125I-insulin binding by rat soleus muscle

Muscle ATP levels were lowered by incubating rat soleus muscles under anaerobic conditions, or in the presence of 2:4-dinitrophenol (0.5 mM), EDTA (5 mM) or mannitol (400 mM). 125I-insulin binding, measured under equilibrium conditions at 25 degrees C, was reduced by 49-71% in ATP-depleted muscles. Insulin binding was also determined using two other procedures which minimized internalization of 125I-insulin: these were (a) 5 min at 25 degrees C, and (b) 24 h at 3 degrees C. Under these conditions, 125I-insulin binding was reduced by 28-55% in ATP-depleted muscles. These results confirm that in soleus muscle the effect of ATP-depletion on 125I-insulin binding is actually concerned with the binding step itself and not merely a reflection of ATP-dependent internalization of the bound hormone.     

Human erythrocytes inhibit complement-mediated solubilization of immune complexes

Incubation of precipitable immune complexes (IC) with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed human E were added to human serum or guinea pig serum, binding of IC to the E occurred and IC solubilization was significantly inhibited. By contrast, SRBC did not bind IC nor inhibit IC solubilization. Because IC binding to human E is mediated by CR type 1 (CR1) we evaluated whether CR1 was responsible for the inhibition of IC solubilization. Human E were treated with trypsin or anti-CR1 mAb. Both treatments abrogated IC binding to human E but did not affect the ability of the human E to inhibit IC solubilization. Human E inhibited C activation by IC. Thus, incubation of IC in human serum caused significant activation of C3 and C5, but not C4. However, when IC were incubated in whole blood or with isolated human E and serum, C3 activation by IC was inhibited significantly. In addition, we demonstrated that the C3b generated during C activation by IC deposited on both IC and human E. Thus, human E may compete for nascent C3 generated during C activation by IC. In conclusion, human E inhibit both complement-mediated solubilization of IC and C activation by IC.     

Capping of immune complexes by sporozoites of Eimeria tenella

Sporozoites of Eimeria tenella were incubated for 10, 20, or 30 min with parasite-specific monoclonal IgG antibody 3D3II from mice and then rinsed in a Tris-buffered glucose saline solution (TBGS). Some sporozoites were then incubated for 10, 20, or 30 min with ferritin- or colloidal gold-conjugated goat anti-mouse IgG antibody and then fixed in 2.5% glutaraldehyde and prepared for transmission (TEM) or scanning (SEM) electron microscopy. Other sporozoites that had been previously exposed to monoclonal antibody were prefixed with 0.25% glutaraldehyde, incubated with ferritin- or colloidal gold-conjugated anti-mouse IgG antibody and then fixed and prepared for TEM or SEM. Control preparations consisted of sporozoites exposed only to TBGS, monoclonal antibody 3D3II or to ferritin- or colloidal gold-conjugated anti-mouse IgG antibody. Capping of immune complexes occurred only on the surface of those sporozoites exposed to monoclonal antibody 3D3II followed by ferritin- or gold-conjugated antibody. Immune complexes moved laterally and posteriorly on the outer surface of the parasite plasma membrane to form a cap at the posterior end of the sporozoite. Capping did not occur in TBGS controls nor in sporozoites treated with monoclonal antibody 3D3II and prefixed in 0.25% glutaraldehyde before exposure to ferritin- or gold-conjugated antibody. Thus, capping of surface antigens did not occur in the presence of monoclonal 3D3II antibody only, whereas specimens exposed to both monoclonal and ferritin- or colloidal gold-conjugated antibodies were able to cap immune complexes.