Complement fixation by solid phase immune complexes. Reduced capacity in SLE sera

We describe an ELISA for assessment of complement function based on the capacity of serum to support fixation of complement components to solid phase immune complexes (IC). Microplates were coated with aggregated bovine serum albumin (BSA) followed by rabbit anti-BSA IgG. The solid phase IC were reacted with human serum. The uptake of C3b, C4b and properdin was measured using biotinylated F(ab)2 antibodies to each of the proteins, avidin alkaline phosphatase, and paranitrophenyl phosphate. Serial samples obtained from 15 patients with systemic lupus erythematosus were investigated. Out of 72 sera, 24 showed a reduced capacity to support incorporation of C4b into solid phase IC. Thirty-one of the sera showed low C3b binding and 59 of the sera a reduced uptake of properdin. The incorporation into solid phase IC of C3b and C4b as well as of C3b and properdin were closely correlated at high disease activity. In general, patients with severe disease manifestations showed low values in the uptake assays. Judging from the results obtained by analysis of serial samples, the uptake of C3b, C4b and properdin, complement mediated solubilization of fluid phase IC and the concentrations of C1q binding IC were useful indicators of disease activity in the patients. The concentrations of circulating C4, C3 and properdin varied less consistently according to disease activity. The concentrations of serum properdin were never found to be low, which was in contrast to the finding of reduced properdin uptake by solid phase IC in most of the samples.     

The disappearance of enzyme-inhibitor complexes from the circulation of man.

1. Complexes of human trypsin and human granulocyte elastase with alpha1-anti-trypsin and alpha2-macroglobulin were isolated and injected intravenously into human volunteers. 2. The elimination of alpha2-macroglobulin complexes with trypsin and elastase followed single-exponential functions with half-lives of 9 and 12 min respectively. The complexes showed no tendency to dissociate. 3. Complexes of alpha1-anti-trypsin with trypsin persisted in the circulation much longer, with a half-life of 3-5 h; complexes of alpha1-anti-trypsin with elastase had an intermediate half-life of 1 h. 4. Dissociation was observed of alpha1-anti-trypsin complexes with transfer of trypsin and elastase to alpha2-macroglobulin. 5. Dialysable radioactivity appeared in the urine soon after the injection of alpha2-macroglobulin complexes, which suggested a breakdown of complexes by cells in the reticuloendothelial system. Radioactivity over the liver achieved maximum values within 30-40 min after the injection of alpha2-macroglobulin complexes but not until 50-70 min after the injection of alpha1-anti-trypsin comlexes. 6. These results support the concept of a key position for alpha2-macroglobulin in the protective mechanisms against endogenous proteases.     

Studies on antigen-antibody complexes. I. Elimination of soluble complexes from rabbit circulation

Solid phase immunoadsorbents were prepared by coupling antigens to agarose. With this technique specific antibodies were easily isolated in large amounts. The γG-globulin class of antibodies isolated in this manner were not denatured as judged by their normal biological half-life in rabbits. Soluble immune complexes at fivefold antigen excess were prepared from isolated specific antibodies and HSA, human λ-chains, human λG-globulins, and a Waldenström''s macroglobulin as antigens. In all these preparations a characteristic immune complex was encountered that represented the smallest stable antigen-antibody union. In the HSA-anti-HSA system they were found to be AgAb₂ complexes, and Ag₂Ab complexes in the γG-anti-γG system. These stable complexes fixed complement ineffectively. Also, a spectrum of larger complexes was present in each system, and these complexes fixed complement effectively. With intact antibodies the disappearance curves of immune complexes from the circulation were composed of three exponential components. The immune complexes larger than AgAb₂ were quickly removed from the circulation with half-lives of 0.09–0.37 hr. Their clearance was not dependent on complement components, in that depletion of complement by cobra venom factor and aggregated γG-globulin did not alter the pattern of their removal from the circulation. However, when the interchain disulfide bonds of antibodies were reduced and alkylated, the removal of the λ-anti-λ, HSA-anti-HSA, and γG-anti-γG complexes was altered. In these experiments the disappearance curves were composed of two exponential components and the rapid removal of the greater than AgAb₂ complexes did not occur. The immune complexes prepared from reduced and alkylated antibodies fixed complement ineffectively. The presented data indicate that the rapid removal of circulating immune complexes, containing γG-globulin molecules as antibodies, depends primarily on the number of antibodies involved. Furthermore, complement fixation is not involved in the rapid removal of such complexes. Nevertheless, the rapid removal of immune complexes and their ability to fix complement have similarities for optimal function in that both processes require intact interchain disulfide bonds of antibodies and complexes that exceed the AgAb₂ combination.     

Role of fibronectin on the clearance and tissue uptake of antigen and immune complexes in rats.

In the present study, we have evaluated how plasma fibronectin (FN) and tissue FN can affect the clearance from the circulation and organ uptake of antigen or immune complexes (IC) that have the capacity to bind to FN. Phenylated gelatin (DNP-GL) (a FN binding antigen) and IC composed of DNP-GL and monoclonal IgGl anti-dinitrophenol (DNP) antibodies were tested. These probes were compared with DNP-bovine serum albumin (BSA) (a non-FN-binding antigen) and DNP-BSA IC formed with the same anti-DNP antibody used for the preparation of DNP-GL IC. We found evidence that DNP-GL, but not DNP-BSA, formed complexes with soluble FN in vitro and the data strongly suggest that DNP-GL-FN complexes form in vivo. The formation of complexes with plasma FN aided in the clearance of DNP-GL from the circulation, as shown by the facts that DNP-GL was removed from the circulation much faster than DNP-BSA and that complexes of DNP-GL with plasma FN were removed from the circulation faster than uncomplexed DNP-GL. The sites of deposition of DNP-GL were also different from those of DNP-BSA. Thus, DNP-GL demonstrated higher hepatic, splenic, and renal uptake than did DNP-BSA. Renal uptake of DNP-GL was quite high despite the fact that DNP-GL is anionic. Indeed, expressed per gram of tissue, liver and kidney deposition of DNP-GL was not significantly different. By immunofluorescence microscopy, DNP-GL could be demonstrated in hepatic sinusoids and glomerular mesangium. In vitro, DNP-GL bound to FN in the mesangium of frozen sections of kidney tissue. IC formed with DNP-GL or DNP-BSA demonstrated virtually the same size, yet the fate of DNP-GL IC was strikingly different from that of DNP-BSA IC. The removal of DNP-GL IC from the circulation was mediated by the antigen and not by Fc receptors since gelatin (an inhibitor of DNP-GL clearance) but not aggregated IgG (an inhibitor of Fc receptors) inhibited the removal of DNP-GL IC from the circulation. In summary, these studies suggest that the ability of an antigen or IC to bind to FN markedly influences the fate of that antigen or IC. Specifically, binding to FN accelerates clearance from the circulation and favors hepatic and renal (primarily mesangial) uptake of the FN binding antigen of IC.     

Isolation of circulating immune complexes using Raji cells. Separation of antigens from immune complexes and production of antiserum.

Raji cells were used for the isolation of complement-fixing antigen-antibody complexes from serum. Immune complexes bound to these cells were radiolabeled at the cell surface with lactoperoxidase. The complexes were then eluted from the cells with isotonic citrate buffer pH 3.2 or recovered by immunoprecipitation of cell lysates. The antigen and antibody moieties of the complexes were isolated by dissociating sucrose density gradient centrifugation or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of preformed immune complexes were successfully isolated from serum with this approach. In addition, these techniques were used to isolate and identify the antigens in immune complexes in the serum of rabbits with chronic serum sickness and rats with Moloney virus-induced sarcomas. Methods were also developed for the production of antisera against the antigenic moiety of immune complexes isolated from serum. Repeated challenge of rabbits with whole Raji cells with bound complexes or eluates from such cells resulted in antibody production against the antigens of the immune complexes, although reactivity against cellular and serum components was also elicited. Monospecific antisera against the antigens in immune complexes were produced by immunizing rabbits with the alum-precipitated antigen isolated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These techniques may be useful in isolating antigens in immune complex-associated diseases of unknown etiology.     

The clearance time of infused hematopoietic stem cell from the blood circulation

There is no detailed information about the clearance time of infused hematopoietic stem cell (HSC) from the blood circulation in humans. In this prospective study, peripheral blood CD34+ cell counts were detected during the 4 days period following autologous HSC transplantation in 20 patients by means of flow cytometry. The median CD34+ cells were at the highest level in the first hour and decreased below pre-infusion values on the first day after HSC infusion. By nonparametric analysis, positive correlation was found between CD34+ cell levels at the first hour and the post-thaw CD34+ cell dose (r = 0.57, p = 0.01). An inverse correlation was determined between CD34+ cell levels at the first hour and neutrophil engraftment (r = ?0.54, p = 0.01). Compared with the patients having CD34+ cell count of ?2 μL^?1 in the first hour following HSC infusion, the patients having CD34+ cell count of <2 μL^?1 had delayed both neutrophil (20 vs. 12, p = 0.008) and platelet (47 vs. 11, p = 0.01) engraftments. Our results indicated that infused HSCs were removed from the blood circulation within 1 day. In addition, CD34+ cell levels at the first hour may be used as an important indicator to predict the delay of neutrophil and platelet engraftments.     

The effect of complement depletion on lung clearance of bacteria.

We have investigated the effect of hypocomplementemia on early pulmonary clearance of four species of bacteria. The experiments were performed in an inbred animal model to minimize immunologic variability. Complement was depleted by cobra venom factor, and activity in serum was monitored with a phagocytic assay. Bacterial specific antibodies were examined by an indirect radioimmunoassay, and animals with high levels of activity were excluded from anaysis. 4 h after aerosolization with Streptococcus pneumoniae, complement-depleted animals had cleared only 75% of the initial number of organisms, whereas saline-treated controls cleared 91% (P less than 0.01). Aerosolization with Pseudomonas aeruginosa was followed at 4 h by a twofold greater growth of organisms in the complement-depleted animals (446% of original deposition) as compared to the saline-treated controls (211% of original deposition) (P less than 0.02). Clearance of Klebsiella pneumoniae and Staphylococcus aureus were similar in complement-depleted animals and saline-treated controls. These experiments suggest that hypocomplementemia predisposes to bacterial pneumonia and may explain the high incidence of pulmonary infections in patients having impaired complement activity. Our results further indicate that varying defense mechanisms may be involved with clearing the lung of differing bacterial species.     

Intravenous infusion of apolipoprotein E accelerates clearance of plasma lipoproteins in rabbits.

Plasma cholesterol levels in cholesterol-fed rabbits were markedly reduced by the intravenous infusion or bolus injection of recombinant human apo E or rabbit plasma apo E. Administration of 6-70 mg of apo E resulted in an approximately 20-40% acute reduction in plasma cholesterol levels within 2-3 h. Plasma cholesterol levels remained reduced for 4-8 h after the administration of apo E. Furthermore, the intravenous injection of apo E reduced the plasma cholesterol levels in Watanabe heritable hyperlipidemic rabbits. The addition of apo E to [14C]cholesterol-labeled canine thoracic duct lymph or [14C]cholesterol-labeled chylomicrons resulted in accelerated plasma clearance of these diet-induced lipoproteins in normal rabbits, with the uptake occurring primarily in the liver. This study suggests that the amount or availability of apo E in the plasma of cholesterol-fed rabbits may be rate limiting for the normal clearance of diet-induced remnant lipoproteins.     

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

The possible role of group specific component (Gc) (vitamin D-binding protein) in the clearance of cellular actin entering the circulation was examined with 125I-labeled Gc and actin injected into a rabbit model. Although filamentous F-actin is depolymerized primarily by plasma gelsolin, greater than or equal to 90% 125I-actin injected in either monomeric G- or F-form became complexed eventually with Gc (1:1 molar ratio). Clearance of Gc complexes was much faster (greater than 90% within 5 h) than that of native Gc (t1/2 = 17.2 h). Nephrectomy did not significantly alter the clearance of either Gc or actin. Since Gc complexes are dramatically increased in situations of tissue necrosis such as in fulminant hepatic failure, the current results suggest a crucial role for Gc in sequestration and clearance of released cellular actin.     

Analysis of sera from ovarian cancer patients for immune complexes

In contrast to other malignancies, circulating immune complexes (CICS) are usually not detected by conventional assays in the sera of ovarian cancer patients. However, a polyethylene glycol (PEG) precipitation assay has been reported to detect putative CICS in ovarian cancer. To determine if CICS were indeed present, we analyzed sera from 12 women with ovarian cancer. All were negative for CICS by the Raji cell assay; 5 (42%) were positive by the PEG assay. However, the PEG precipitate did not possess characteristics of immune complexes. IgG in sera or in the precipitate sedimented in sucrose gradients solely at the same rate as 7S monomeric IgG. In addition, the precipitates were not able to activate the complement system and the four IgG subclasses were present in the same relative concentration as that found in normal serum. The results suggest that it is probably a misnomer to label the material detected in ovarian cancer sera by the PEG precipitation assay as CICS. Instead a non-immune interaction of IgG with other components, possibly membrane fragments, is probably being measured.     

Demonstration of cryoprecipitable immune complexes in pneumococcal pneumonia.

Cold-insoluble protein complexes (cryoprecipitates) can be found in the serum in a variety of infectious diseases. We studied serum cryoprecipitates isolated from three patients with pneumococcal pneumonia by counterimmunoelectrophoresis (CEP) and immunofluorescent technics for the presence of immune complexes. The cryoprecipitates and supernatant serum were tested for pneumococcal capsular polysaccharide (PCP) by CEP at 37 C and 56 C with the appropriate controls. Antibodies against PCP in the cryoprecipitates and the supernatant serum were detected as follows. Streptococcus pneumoniae from each case was fixed onto slides. The slides were incubated with each cryoprecipitate and supernatant serum at 37 C, and further incubated with fluorescein isothiocyanate-conjugated antisera to human IgG, IgM, and IgA. The slides were examined with an immunofluorescent microscope. PCP was demonstrated in all of the cryoprecipitates. IgG antibodies against PCP were detected in all of the cryoprecipitates, while IgM antibodies were detected in Cases 1 and 2, and IgA antibodies in Case 1 only. Complement components of C3 and C4 also were demonstrated in the cryoprecipitates by CEP. These findings suggest that some patients with pneumococcal pneumonia have cryoprecipitable-immune complexes consisting of PCP and its antibodies.     

Hepatic glutathione depletion and impaired bromosulphthalein clearance early after paracetamol overdose in man and the rat.

1. Plasma clearance of bromosulphthalein was impaired in patients within 8 h, and in rats within 2 h , of paracetamol overdose, before biochemical signs of liver damage had appeared. 2. Paracetamol and bromosulphthalein competed for uptake into the liver and excretion into the bile. Impaired hepatic uptake of bromosulphthalein could also be demonstrated in man at doses of the drug within the therapeutic range. 3. In patients studied a smaller proportion of bromosulphthalein was retained in the plasma as the glutathione conjugate after an overdose than after therapeutic doses. This effect could be reproduced in the rat and shown to be due to depletion of hepatic glutathione and to impairment in the activity of the enzyme glutathione-S-aryl transferase. 4. These studies provide further evidence that depletion of hepatic glutathione occurs after paracetamol overdose in man, as in the experimental animal, allowing the subsequent accumulation and binding of a toxic metabolite of the drug within liver cells. Impaired enzymic conjugation of the toxic metabolite with hepatic reduced glutathione may also be important in this situation.     

Blockade of clearance of immune complexes by an anti-F(cγ) receptor monoclonal antibody

Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcγR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.     

Circulating immune complexes and the immune system in hemorrhagic vasculitis

The paper is concerned with the results of studying the concentration of circulating immune complexes and immune system (the number of lymphocyte populations and subpopulations in the peripheral blood, the level of serum immunoglobulins) in 45 patients aged 10 to 72 years with hemorrhagic vasculitis depending on the disease pattern and course before and after the treatment. The patients suffering from hemorrhagic vasculitis manifested appreciable changes in humoral immunity and negligible derangement of cellular immunity associated with dramatic enhancement of complex formation. The intensity of the alterations was discovered to be dependent on the pattern and course of the disease.