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.     

Microparticles released by human neutrophils adhere to erythrocytes in the presence of complement

The release of cell surface-derived microparticles, or ectosomes, has now been described for many different cell types. In various diseases characterized by systemic inflammation, the numbers of ectosomes released from specific cell-types are found increased manifold in the circulation. Their pro-inflammatory and pro-coagulant functions make them potentially important actors in disease establishment and/or progression. Until now, ectosomes have been believed to be free in the circulation. Herein, we provide evidence for sequestration of ectosomes derived from human polymorphonuclear neutrophils to erythrocytes, similarly to immune complexes. We show that ectosomes activate and bind complement in vitro. In whole blood, opsonization of ectosomes by complement mediated their immune adherence to erythrocytes through complement receptor 1. Taken together, our data suggest an important role for complement and erythrocytes in the sequestration, and possibly clearance, of blood-borne ectosomes stemming from neutrophils. The immune adherence described here may modify the biological activity and function of ectosomes.     

The kinetics of clearance of human antibody/dsDNA immune complexes from rabbit circulation. Effects of dsDNA size, rheumatoid factor, and reduction and alkylation of the antibodies

We have conducted in vivo investigations of the rate of clearance of prepared antibody/dsDNA immune complexes from the circulation of rabbits and have correlated the results with some in vitro immunologic properties of the complexes. Those complexes that fix complement most efficiently in vitro (as manifested by their complement-mediated binding to human red blood cells or rabbit platelets) are cleared most rapidly. Factors that decrease this binding such as reduction and alkylation of the antibodies or the use of dsDNA of a lower m.w. also tend to prolong the circulation times for the complexes. This study provides additional evidence in support of a complement-mediated platelet immune complex clearance mechanism in non-primates that parallels the erythrocyte immune complex clearance mechanism in primates first described by He bert. Finally, the implications of our findings with respect to the importance of dsDNA size in the pathogenesis of systemic lupus erythematosus are discussed.     

Abnormal immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with AIDS.

C3b-coated immune complexes (IC) adhere to complement receptor 1 (CR1) on human E in the circulation. E from AIDS patients have an acquired low CR1 number. To study immune adherence and IC elimination in AIDS, radiolabeled hepatitis B surface Ag/antibody complexes were injected i.v. in six AIDS patients and in 14 healthy controls. The binding of IC to E was reduced in AIDS patients (mean binding 2 min after injection: 24.9 +/- 13.3%) compared with healthy individuals (63 +/- 3.7%) (p = 0.0005). The low binding correlated directly with the number of CR1/E and to the capacity of these E to bind IC in vitro. During the first 15 min disappearance of IC was faster in AIDS patients than in normal subjects and correlated with CR1 number. Thereafter, elimination was very slow in AIDS patients, which suggested that a fraction of IC might be released back into the circulation similarly to what has been observed for C3b-coated E. When the data were analyzed with a mathematical model allowing for such release to occur, five of six AIDS patients had a high release rate compared with little or no release in normal individuals (p less than 0.001). Thus, low CR1 on E is responsible for defective immune adherence, and might determine abnormal disappearance of IC from the circulation as well.     

Clearance of blood-borne pathogens mediated through bispecific monoclonal antibodies bound to the primate erythrocyte complement receptor

 The primate erythrocyte complement receptor facilitates both the immune adherence reaction and the immune complex clearance properties of primate erythrocytes. These phenomena have been studied for more than 40 years. However, it has only recently become apparent that these characteristics of primate erythrocytes may be useful in the generation of a therapy based on bispecific monoclonal antibodies. Our approach uses bispecific monoclonal antibody constructs (heteropolymers) that promote binding of specific target pathogens to primate erythrocytes via the complement receptor. Once bound to the erythrocytes, the pathogen-heteropolymer complex should be cleared from the circulation, phagocytosed and destroyed in the liver. Results with several prototype target pathogens in monkey models indicate it may be possible to use this technology to develop a robust and general therapy for the treatment of diseases associated with blood-borne pathogens. Accepted: 14 October 1997     

Molecular bases of immune complex pathology

The binding of antigens with antibodies forms immune complexes in the body. Usually these complexes are eliminated by the system of mononuclear phagocytes without development of pathological changes. This review highlights principal mechanisms responsible for safe removal of immune complexes in primates and humans. Special attention is given to diseases known as “immune complex diseases”, when antigen-antibody complexes induce inflammatory reactions. The review considers key experimental works that significantly contributed to current knowledge of etiology and pathogenesis of type III hypersensitivity. Some factors of the development of immune complex syndrome such as level of humoral immune response to antigen, isotype and affinity of forming antibodies, the amount of immune complexes, and the consequences of their interaction with the complement system and Fc-receptors are analyzed based on the molecular mechanisms involved. The review contains a retrospective analysis of the most significant scientific achievements in immune complex pathology investigation within the last 100 years.     

The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans. Complement- and erythrocyte complement receptor 1-dependent mechanisms

The role of complement and its receptor on erythrocytes (CR1) in the physiologic elimination of large immune complexes from the circulation of humans was assessed. Large radiolabeled soluble tetanus toxoid- anti-tetanus toxoid complexes were injected i.v. into three normal individuals and three patients with SLE. These complexes were prepared in antibody excess and were 45S in size, fixed C and bound to E CR1 in vitro. The percentage of complexes bound in vitro was directly proportional to CR1 number/E in four normal subjects and three SLE patients. After i.v. injection into normal subjects, complexes were cleared rapidly, with a monoexponential rate constant (10.3 to 11% complexes cleared/min). In the SLE patients, clearance was best explained by two phases: the first occurred within the first minute indicating immediate trapping of a fraction of the complexes (19.5 to 25.3% of injected complexes trapped), the second was monoexponential and was similar to the normal range. A large fraction of complexes bound within the first minute to E in vivo; the percentage of binding was variable, ranging from 16.3% to 71.5% and was related to E CR1 number. In a second study complexes were injected that had been attached to autologous E by opsonization with C in vitro. Their elimination was similarly monoexponential, except in one SLE patient in whom there was significant initial trapping (30.9%). A fraction of these complexes were released from E within the first minute, the percentage release being greatest in the patient with the lowest CR1 number (81.4%). E bearing immune complexes remained in the circulation and were not transiently sequestered in the liver or spleen. This is the first study of the clearance of soluble immune complexes in vivo in humans and shows that C and CR1 on E participate in immune complex clearance reactions, and that abnormal clearance can be detected in the form of rapid removal of immune complexes from the circulation.     

Prognostic value of assays for circulating immune complexes and natural cytotoxicity in malignant skin melanoma (stages I and II)

Summary The aim of the present study was to determine the possible prognostic value of immune complex determinations and estimations of natural cytotoxicity in melanoma patients. Circulating immune complexes were assayed in 46 patients suffering from malignant skin melanoma (stages I and II) by a direct complement consumption (CC) test, a polyethylene glycol (PEG) precipitation CC assay and a solid-phase Clq-protein A-binding assay. The presence of both IgG- and IgM-containing complexes in IC-positive melanoma sera was confirmed by an Ig-class-specific precipitation-immunoradiometric assay. Results based on this assay and reduction-alkylation data indicated that IgM-containing IC were responsible for a major part of the IC activity recorded in the CC-assays.Soluble immune complex activity, as measured by the direct CC test and the PEG-CC assay, correlated with relapse, whereas the expected inverse relationship between natural cytotoxicity and recurrence was not established. This finding may be explained by the apparent predominance of IgM-containing immune complexes in patients subsequently showing relapse.     

C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion.

Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.     

Platelet immune complex interaction in pathogenesis of Kawasaki disease and childhood polyarteritis.

The role of platelets in the pathogenesis of vasculitis and the formation of coronary artery aneurysms was studied in 19 children with Kawasaki disease and five with polyarteritis. All patients with Kawasaki disease developed thrombocytosis in the third week of illness. The peak platelet count was significantly correlated (p less than 0.005) with the subsequent development of coronary artery aneurysms. The rise in platelet count was associated with the appearance in the circulation of a factor that induced aggregation and serotonin release in normal platelets. This factor was shown to be of high molecular weight, and its activity was lost at low pH--features suggestive of an immune complex. Immune complexes, detected by precipitation with polyethylene glycol, also appeared in the circulation as the platelet count increased. These complexes induced platelet aggregation, and there was a significant correlation (p less than 0.001) between the concentrations of IgG and IgA in the polyethylene glycol precipitated material and the platelet aggregating activity. Similar platelet aggregating activity was also detected in patients with polyarteritis but followed a different time course, persisting in the circulation for several months in association with continued disease activity. These findings imply that different mechanisms have a role in distinct phases of Kawasaki disease. The initial feverish phase (probably infective) is probably followed by an immune complex vasculitis that occurs when antibodies to the initiating agent appear in the circulation. The immune complexes aggregate platelets and induce release of serotonin. Platelet derived vasoactive mediators may increase vascular permeability and facilitate further deposition of complexes in the tissues.     

An abnormality of immune complex kinetics in murine lupus

In order to understand better the role of immune complex metabolism in the pathogenesis of autoimmune diseases, we have investigated the early stages of immune complex uptake by the liver, the major organ responsible for clearance of soluble complexes in the mouse. Livers were perfused in situ via the portal vein over 3 to 5 min with trace amounts of radiolabeled soluble model immune complexes. In 4 nonautoimmune strains (BALB/c, DBA/2, CAF1, NZW) 60 to 72% of the model complexes perfused were taken up and remained in the liver after 20 min of continuous perfusion with oxygenated Krebs-Henseleit buffer. In NZB and NZB/W F1 female mice at ages 0.5 to 11 mo, 66 to 78% of the model complexes remined in the liver. However, when a dose of heat-aggregated human gamma-globulin sufficient to saturate the reticuloendothelial system was perfused 7 min after radiolabeled complexes, 15.2 +/- 7.2% (mean +/- SD) of the complexes were displaced in the nonautoimmune strains. In contrast, 32.6 +/- 10.5% of the complexes were displaced from the liver in NZB and NZB/W F1 female mice (p < 0.001). Thus, although hepatic uptake of immune complexes in autoimmune mice appears to be normal or even enhanced, there may be impaired phagocytosis by the hepatic RES or weaker binding of complexes to the surface of the Kupffer cells. Such surface-bound immune complexes remaining accessible to the circulation may contribute to the autoimmune process.     

Inhibition of macrophage tumoricidal activity by immune complexes and altered erythrocytes

Engagement of the macrophage membrane by biologic particles including insoluble immune complexes inhibited the development of lymphokine-mediated nonspecific tumoricidal activity by murine macrophages. The degree of inhibition was dependent on the dose of particles and the lymphokine concentration. Inhibition was not due to macrophage cell death or to diminution of cell adherence after ingestion of the immune complexes. Soluble immune complexes were not inhibitory, although approximately 10% of the complexes became cell-associated. Monomeric or heat-aggregated IgG was also not inhibitory. IgG-opsonized erythrocytes (EA) were inhibitory and inhibition was dependent on the degree of opsonization. In contrast, nonopsonized erythrocytes (E), which did not bind to macrophages, were not inhibitory. Phagocytosis of glutaraldehyde-treated E or E carrying IgM antibody and complement (EAC) also led to a reduction of tumorilytic activity. Insoluble immune complexes were inhibitory when added either before or after lymphokine. Phagocytosis was neither sufficient nor necessary to cause inhibition because 1) ingestion of polystyrene latex beads did not diminish tumoricidal activity, and 2) macrophages plated on IgG-coated surfaces were inhibited with respect to the tumoricidal function. Inhibition was not affected when indomethacin (10(-6) M) was included in the assay, which indicated that prostaglandins were not involved in the process. Thus, macrophage tumoricidal responsiveness may be compromised by interaction of biologic substances with macrophage plasma membranes. This process may thereby inactivate an important host defense mechanism against neoplastic cells.     

Hereditary C2 deficiency associated with immune complex disease.

A patient presenting with a syndrome probably due to immune complex deposition was investigated and found to possess an inherited C2 complement deficiency. Family studies indicated that the deficiency was transmitted as an autosomal recessive trait. HLA typing for the HLA-A and HLA-B specificities and HLA-D specificities indicated a close linkage between the HLA and C2 genes, as has been described elsewhere. The HLA-A and B locus specificities HLA-AW25 and HLA-B18 were coded for by each of the two chromosomes carrying the C2(0) gene. However, the two chromosomes differed at the HLA-D locus, as one coded for HLA-DW2 whilst the other did not. This case, therefore, provides a unique haplotype and may be of importance in mapping the C2(0) locus, as it suggests that the gene order on chromosome 6 is HLA-D, C2(0), HLA-B, HLA-A. Extensive complement component assays indicated that utilization of complement in the patient was occurring via the alternate complement pathway. It is suggested that, as a result of the C2 deficiency, infections with viruses and other agents could lead to an immune complex disease due to an impaired capacity to effectively eliminate circulating complexes.     

A study of in vivo immune complex formation and clearance in man

C and CR1 have been shown to participate in the clearance of injected, preformed, immune complexes in humans and in non-human primates. Their role in the physiologic disposal of immune complexes formed in vivo in humans was investigated in three patients receiving radioimmunotherapy for ovarian carcinoma. On day 0 each patient received, by intraperitoneal injection, 10 mg of 131I-mouse anti-tumor mAb (10 mCi/mg). On days 1 and 2, 18 mg of trace-labeled, 125I-human anti-mouse IgG was administered by i.v. infusion over 15 min, to accelerate the clearance of the 131I-anti-tumor antibody from the circulation and reduce the radiation dose to the marrow. Sequential blood samples were obtained after the injection of the second (anti-mouse) antibody, to monitor clearance. Immune complexes (shown by sucrose gradient centrifugation to be 19 to 40 S in size) formed within 5 min, and were cleared with a half-life of 11 +/- 1.7 min in the liver. Complexes were measured by 4% polyethylene glycol precipitation, and by solid phase C3d- and C1q-binding assays. Between 8 and 11% of the total available complexed material bound to CR1 on E. Peak binding of immune complexes to red cells occurred 10 min after the maximal complex load was detected by precipitation with polyethylene glycol. At that time, immune complexes bound to E constituted one-fifth of the total circulating pool of complexes. Coincident with immune complex formation and clearance, a 47% fall in serum C4, C3, and CH50 was measured, with the deposition of up to 1230 molecules of C4, and 2590 molecules of C3 on the surface of red cells. During 20 min after immune complex formation there was a mean loss of 32% of erythrocyte CR1. The changes in complement and CR1 on E and in serum observed in these patients resembled those seen in patients with SLE: i.e., a reduction in CR1 and an increase in C3 and C4 on E, and reduced serum C.     

Complement-mediated adherence of immune complexes to human erythrocytes. Difference in the requirements for C4A and C4B

The classical pathway of complement is required for the adherence of soluble tetanus toxoid (TT)-human anti-TT complexes to erythrocytes. Using human C4-deficient serum we compared the capacity of the two forms of human C4 (C4A and C4B) to mediate this function: C4A was shown to be 1.5-fold more efficient than C4B. In contrast, haemolysis by C4B was 3.7-fold more efficient than by C4A. Such large differences suggest that both forms are complementary, and that C4A is preferentially involved in the processing of immune complexes in humans.     

Langerhans cells: target cells in immune complex reactions.

Skin of normal, cobra venom extract-treated, and C₄-deficient guinea pigs was injected with ferritin-antiferritin or with peroxidase-antiperoxidase immune complexes. Skin and draining lymph nodes were studied to compare the phagocytosis of these immune complexes by Langerhans cells (LC) and by macrophages. When complement was present, immune complexes were damaging to LC, and uptake of the immune complexes, although present, was limited. When components of complement were absent or diminished, increased numbers of LC in lymph nodes were seen, but damage to LC was absent or decreased. However no detectable change in the amount of phagocytosis by LC was noted. Since some LC can carry antigen from skin to lymph nodes and may be involved in the presentation of antigen to lymphoid cells in some cell-mediated immune responses, impairment or abolition of LC function by immune complexes could represent a mechanism through which the local presence of antibodies might interfere with the induction and elicitation of cellular immunity by antigen. Moreover, damage to LC and subsequent release of intracellular (lysosomal?) substances may constitute a general mechanism of response in the skin to injury and may be an integral part of inflammatory and allergic skin reactions.     

Mononuclear phagocyte system in SLE. II. A kinetic model of immune complex handling in systemic lupus erythematosus

Using the principles of reaction kinetics, we constructed a model for the handling of immune complexes and the pathogenesis of SLE immune complex disease. The model incorporates rate constants for complement- and Fc-mediated clearance, parameters for autoantibody, complement and immune complex levels, and scores for clinical disease activity. The model assumes that complement fixation by immune complexes is a prerequisite for complement-mediated clearance and that disease activity results from immune complex deposition. To test the relationships derived, data from 32 lupus patients were analyzed and the predictions were compared with actual findings. The model predicts a low correlation coefficient between disease activity and immune complex levels (found, r = 0.25, p greater than 0.1). The model also predicts a poor correlation between disease activity and impaired Fc-mediated clearance in patients with normal complement levels (found, r = 0.10, p greater than 0.1), but a high correlation coefficient between disease activity and impaired Fc-mediated clearance in patients with hypocomplementemia (found, r = 0.61, p less than 0.001). In patients with normal complement levels, the model predicts a good correlation between anti-DNA antibody and immune complex levels (found, r = 0.71, p less than 0.001), whereas hypocomplementemic patients should have a good correlation between anti-DNA to CH50 ratios and immune complex levels (found, r = 0.73, p less than 0.001). The model predicts that disease activity should correlate better with the product of the anti-DNA to CH50 ratio and the rate constant for Fc-mediated clearance than with any single parameter (found, r = 0.85, p less than 0.0001). These significant correlations, which were predicted by the model, suggest that complement-mediated mechanisms are the first line of host defense against immune complex-induced injury, that the efficiency of complement opsonization plays a central role, and that both abnormal complement- and Fc-receptor function leads to active renal disease in SLE.     

Complement mediates the binding of HIV to erythrocytes

A fraction of HIV is associated with erythrocytes even when the virus becomes undetectable in plasma under antiretroviral therapy. The aim of the present work was to further characterize this association in vitro. We developed an in vitro model to study the factors involved in the adherence of HIV-1 to erythrocytes. Radiolabeled HIV-1 (HIV) and preformed HIV-1/anti-HIV immune complexes (HIV-IC) were opsonized in various human sera, purified using sucrose density gradient ultracentrifugation, and incubated with human erythrocytes. We observed that, when opsonized in normal human serum, not only HIV-IC, but also HIV, bound to erythrocytes, although the adherence of HIV was lower than that of HIV-IC. The adherence was abolished when the complement system was blocked, but was maintained in hypogammaglobulinemic sera. Complement-deficient sera indicated that both pathways of complement were important for optimal adherence. No adherence was seen in C1q-deficient serum, and the adherence of HIV was reduced when the alternative pathway was blocked using anti-factor D Abs. The adherence could be inhibited by an mAb against complement receptor 1. At supraphysiological concentrations, purified C1q mediated the binding of a small fraction of HIV and HIV-IC to erythrocytes. In conclusion, HIV-IC bound to erythrocytes as other types of IC do when exposed to complement. Of particular interest was that HIV alone bound also to erythrocytes in a complement/complement receptor 1-dependent manner. Thus, erythrocytes may not only deliver HIV-IC to organs susceptible to infection, but free HIV as well. This may play a crucial role in the progression of the primary infection.     

Complement-dependent immune complex-induced bronchial inflammation and hyperreactivity

Bronchoconstriction responses in the airway are caused by multiple insults and are the hallmark symptom in asthma. In an acute lung injury model in mice, IgG immune complex deposition elicited severe airway hyperreactivity that peaked by 1 h, was maintained at 4 h, and was resolved by 24 h. The depletion of complement with cobra venom factor (CVF) markedly reduced the hyperreactive airway responses, suggesting that complement played an important role in the response. Blockade of C5a with specific antisera also significantly reduced airway hyperreactivity in this acute lung model. Complement depletion by CVF treatment significantly reduced tumor necrosis factor and histamine levels in bronchoalveolar lavage fluids, correlating with reductions in airway hyperreactivity. To further examine the role of specific complement requirement, we initiated the immune complex response in C5-sufficient and C5-deficient congenic animals. The airway hyperreactivity response was partially reduced in the C5-deficient mice. Complement depletion with CVF attenuated airway hyperreactivity in the C5-sufficient mice but had a lesser effect on the airway hyperreactive response and histamine release in bronchoalveolar lavage fluids in C5-deficient mice. These data indicate that acute lung injury in mice after deposition of IgG immune complexes induced airway hyperreactivity that is C5 and C5a dependent.     

Clearance and organ localization of small DNA anti-DNA immune complexes in mice

DNA anti-DNA immune complexes (IC) play a major role in the pathogenesis of SLE. We studied the clearance and organ localization of small DNA anti-DNA IC formed at different Ag/antibody ratios in normal mice. IC formed at Ag excess, containing areas of "exposed" DNA not covered by IgG, showed rapid Ag-mediated clearance from the circulation by the liver. DNAse digestion of these IC in vitro yielded small IC devoid of exposed DNA that were cleared more slowly from the circulation. IC formed at antibody excess were cleared by an Ag-independent mechanism at rates proportional to the number of IgG in the IC. None of the IC studied bound significantly to complement receptors on circulating cells in vivo or in vitro. For all IC, after initial rapid clearance, 10 to 20% of the injected material persisted in the circulation. Analysis of these IC showed that they were processed in vivo to yield complexes similar to those generated by in vitro DNAse digestion. We conclude that IC containing exposed DNA are removed rapidly from the circulation by Ag-mediated clearance. However, in vivo processing of IC occurs to yield smaller IC that are cleared slowly. We propose that these IC containing small DNA may persist in the circulation and accumulate in tissues, thereby playing an important role in the pathogenesis of tissue injury in SLE.