Circulating immune complexes containing chlamydial lipopolysaccharide in acute myocardial infarction

The presence of circulating immune complexes (IC) was studied using two detection methods specific for chlamydial lipopolysaccharide (LPS) in paired serum samples of 44 patients (30 men and 14 women) with acute myocardial infarction (AMI). Forty-four random controls were individually matched for locality, age and sex with the AMI patients. As specificity controls for the IC assays single serum samples from 29 patients with diseases characterized by the presence of circulating IC were used. Fifty-seven per cent of AMI patients, 12% of their random controls and 10% of the patient controls were shown to have chlamydial LPS-specific immune complexes in their sera (P less than 0.0001, AMI versus random and patient controls). This finding provides further evidence of the possible association of chronic chlamydial infection with AMI.     

Deposition of immune complexes in ovarian follicles of mice with lupus-like syndrome.

The occurrence of immune deposits in the ovaries of mice with lupus-like syndrome was studied by immunofluorescence, light microscopy, and electron microscopy. Granular deposits of mouse IgG and C3, and occasionally gp70 and denatured DNA, were found in the zona pellucida of mature and atretic follicles. Dense deposits of foreign material were seen by light and electron microscopy in areas of ZP corresponding to the immune deposits. These lesions, presumably induced by immune complexes, resemble the "membranous" changes observed in the glomerular basement membrane in some of the same mice. Inflammatory changes of the ovarian follicles were not observed. The study of "membranous" immune complex oophoritis could contribute to the understanding of immunologic mechanisms of the female reproductive system.     

Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A

Evidence for the presence of circulating immune complexes was found in thirty-four out of fifty-five samples from forty-seven patients with haemophilia A. In eleven patients the complexes, precipitated from the blood with polyethylene glycol, were digested with pepsin. The F(ab')2 antibody was tested, and found to have neutralizing activity against coagulant Factor VIII in two patients. In one of these no free antibody had ever been found in the plasma, while in the other the antibody was concentrated tenfold in the complex. In two other samples free without complexed antibody was found. In comparison, IgG-containing complexes were found in nine out of nineteen patients with von Willebrand's disease and no complexes were found in the sera from twelve multi-transfused thalassaemics. PEG precipitation is a useful technique for the preparation of concentrated immune complexes for further study such as antigen identification.     

Characterization of immune complexes containing cytomegalovirus-specific IgM antibodies following a kidney graft.

In order to demonstrate the viral specificity of IgM-containing immune complexes (IgM-CIC) detected by a C1q assay in renal allograft recipients developing a CMV infection, a technique is described allowing: 1) the dissociation of IgM-CIC by action of an acid buffer, and 2) the characterization of the viral specificity of IgM antibodies released by this treatment. This step was performed by ELISA and Western Blot. When technique was applied to the follow-up of a renal allograft recipient developing a recurrent CMV infection within 2 months post-graft, it was found that the IgM-CIC detected on the day of the graft were not CMV-specific, whereas the IgM-CIC detected during the second month after transplantation contained CMV-specific IgM antibodies. These CMV-specific IgM-CIC were detected as early as the urinary viral excretion. It was shown by Western Blot analysis that these IgM antibodies reacted with a 45-47 kDa viral polypeptide which is a viral target for specific humoral response at the early phase of CMV infection.     

Interpretation of the Raji cell assay in sera containing anti-nuclear antibodies and immune complexes.

The Raji cell assay is regarded as a test for the detection and quantitation of immune complexes. It is frequently positive in sera from patients with SLE. We have demonstrated a relationship between Raji cell binding and antibodies to DNA and soluble cellular antigens. In five sera containing high titres of antibodies of known single specificity, most of the Raji cell binding occurred in the 7S IgG fraction where the majority of anti-nuclear antibody was also found. When each of these sera was incubated with its specific antigen, Raji cell binding increased. Subsequent fractionation showed that this binding was in the high molecular weight fraction (greater than 200,000 daltons) and that Raji cell binding and antibody activity were abolished in the 7S fraction. These data confirm that Raji cell bind immune complexes but also indicate that 7S anti-nuclear antibodies may interact directly with Raji cells by an unknown mechanism. Therefore, in sera of patients with anti-nuclear antibodies, binding to Raji cells does not necessarily imply the presence of immune complexes alone.     

Characteristics of immune complexes in myocardial infarct

Nonspecific circulating immune complexes (CIC), different in size and stability, are detectable in the blood sera of patients with myocardial infarction; changed concentration of these complexes appears to be a reflection of the defense response of the body, aimed at homeostasis maintenance. No relationship between cardiac glycosides and CIC levels was revealed in patients with myocardial infarction. A reduction of the level of 'large' CICs by days 21-30 of the condition was observed in the patients treated with anticoagulants from the first day of the disease. A low CIC level in the acute period of the disease is a prognostically unfavorable sign in respect of the outcome of the condition.     

Clearance kinetics of soluble pre-formed immune complexes containing IgM antibodies in normal and decomplemented rabbits.

The clearance kinetics of homologous soluble pre-formed 125I-BSA/IgM anti-BSA immune complexes (IgM . IC) injected into the blood was studied in the rabbit. IgM . IC close to equivalence were cleared with a greater velocity than those in x 5 or x 10 antigen excess. Clearance of BSA specifically bound to antibody, was measured by the Farr assay and found to exhibit an exponential mode of elimination suggesting a selective removal of large over small lattice complexes or free antigen. The role of C3 in the clearance of IgM . IC was examined by treating rabbits with cobra venom factor (CVF). While more variation was noted in CVF-treated rabbits, no statistically significant differences could be detected between C3-depleted and control animals. It is, therefore, concluded that soluble preformed IgM . IC are cleared from the rabbit circulation by a C3b-independent mechanism. This finding for soluble IC is clearly different from the clearance of IgM-coated red cells from the circulation, which has previously shown to be highly C3b-dependent.     

Glomerulonephritis induced by pre-formed immune complexes containing monoclonal antibodies of defined affinity and isotype.

The work presented here represents the first report of the induction of experimental immune complex (IC) disease in mice using monoclonal antibodies (MoAb) derived from somatic cell hybridization. IC were formed using two antigens of either high (DNP19BSA) or low (DNP4BSA) epitope density and five MoAb (four IgGl with varying affinities for the dinitrophenol hapten and one IgM with a similar affinity to that of the lowest affinity IgGl). Circulating levels and sizes of IC were dependent on the affinity of the antibody component of the complex. When antigen of high epitope density was used, the glomerular localization of injected IC was diffuse mesangial for the IgM antibody, focal mesangial for the highest affinity IgG and diffuse, and predominantly capillary for the low affinity IgG antibodies. Subepithelial electron dense deposits were observed only with IC made with the low affinity IgG antibodies. When IC containing antigen of a lower epitope density were injected, localization was only observed with IC made near equivalence. Deposition of these IC was less prominent than that found when IC containing antigen of higher epitope density were injected. The relevance of these findings to the pathogenesis of glomerulonephritis is discussed.     

Circulating immune complexes and kidney lesions following 131I administration in mice with thyroglobulin antibodies

Immune complexes containing thyroglobulin have been described in kidneys of some patients with thyroid disease. We investigated the circulating immune complexes (with the Raji cell radioassay) and the kidney histopathology (by immunofluorescence and electron microscopy) in mice that received radioiodine to release thyroglobulin in the circulation, 2 or 4 weeks after immunization with mouse thyroglobulin in Freund's complete adjuvant. Circulating immune complexes and thyroglobulin, antibodies were found in all mice. Granular deposition of IgG, IgM, C3, and thyroglobulin, mainly in the mesangium but also in the capillary walls of the glomeruli, were observed in most of the mice. These experiments suggest that circulating immune complexes composed of thyroglobulin are responsible for the glomerular lesions. Hyperthyroid patients should be tested for thyroglobulin antibodies before treatment with radioiodine to avoid formation of thyroglobulin-containing circulating immune complexes.     

The disappearance kinetics of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice.

Soluble immune complexes prepared with reduced and alkylated antibodies persisted longer in the circulation than complexes prepared with intact antibodies, when these were administered intravenously to mice. The disappearance of complexes with reduced and alkylated antibodies was delayed in part because the initial phase of vascular permeability was considerably less than that seen following the administration of complexes with intact antibodies. In addition, large complexes with lattice structure of more than two antigen and two antibody molecules persisted longer in the circulation after administration of complexes with reduced and alkylated antibodies than after administration of complexes with intact antibodies. Thus, the concentration of large latticed complexes with reduced and alkylated antibodies was significantly greater than the concentrations of large latticed complexes with intact antobodies at all observed times through 96 hours. The persistence of large latticed complexes with reduced and alkylated antibodies was associated with significantly decreased hepatic localization of complexes with reduced and alkylated antibodies compared to the hepatic localization of complexes with intact antibodies at 1, 4, 12, and 24 hours. The observations indicated that the removal of large latticed complexes from the circulation by the hepatic mononuclear phagocyte system was decreased when reduced and alkylated antibodies were used for the preparation of immune complexes. The persistence of large latticed complexes with reduced and alkylated antibodies in the circulation was associated with enhanced and prolonged presence of glomerular deposits of immune complexes, as reported in the accompanying article (Haakenstad AO, Striker GE, Mannik M: Lab Invest 35:293, 1976.     

The glomerular deposition of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice.

The kidney localization and glomerular deposition of soluble immune complexes in mice were greater and more persistent following the intravenous administration of complexes prepared with reduced and alkylated antibodies than following the administration of complexes prepared with intact antibodies. The increased glomerular deposition following the administration of complexes prepared with reduced and alkylated antibodies was associated with the persistence of circulating complexes composed of more than two antigen and two antibody molecules (Haakenstad AO, Mannik M:Lab Invest 35:283, 1976). The deposition of immune complexes in glomeruli, as detected by immunofluorescence, appeared to precede the detection of mouse C3 in glomerular deposits following the administration of both preparations of complexes. The deposition of mouse C3 was more intense and persisted longer in mice receiving complexes containing reduced and alkylated antibodies than in mice receiving complexes containing intact antibodies. The ultrastructural studies indicated that both preparations of complexes initially localized as electron-dense material in endothelial cell fenestrae and in the subendothelial space of the glomerular capillary loops and subsequently accumulated in the mesangial matrix between mesangial cells. The material persisted in the mesangium of mice receiving complexes with reduced and alkylated antibodies, whereas it was removed from the mesangium of mice receiving complexes with intact antibodies. The mechanism for removal of complexes from the mesangial matrix was not defined, but it did not appear to occur through phagocytosis by the mesangial cell.     

In vivo generation and clearance of soluble immune complexes containing IgM antibodies in normal and decomplemented rabbits.

Soluble immune complexes containing IgM antibodies (IgM.IC) were generated in vivo utilizing a passive induction model, whereby purified antibodies were injected into rabbits with circulating radiolabelled bovine serum albumin (BSA) as antigen. A triphasic response was obtained consisting of an initial rapid elimination of TCA-precipitable antigen in the first 30 min, followed by a progressive diminution in the clearance velocity as antigen from the tissues moved back into the circulation to re-equilibrate, and subsequent elimination of the antigen at a rate close to that of free BSA. The dynamics of IC formation and disappearance were studied by a combination of Farr assay and solid-phase C1q binding. The results show that the rate of clearance decreased as the complexes progressively moved into antigen excess, and that the decrease in the proportion of complexed antigen was mirrored by a similar decrease in the ability of the complexes to bind C1q. Depletion of complement by treatment with cobra venom factor did not inhibit the clearance of the antigen, but may have inhibited solubilization of the complexes in vivo. Tissue localization experiments indicated that the liver is the organ predominantly involved in the uptake and catabolism of in vivo-generated IgM.IC. These results show that the clearance velocity of soluble IgM.IC is critically dependent on the antigen/antibody ratio, and that clearance is mediated via a C3b-independent mechanism in the RES.     

Soluble immune complexes in lupus mice: clearance from blood and estimation of formation rates

Elevated levels of circulating immune complexes (IC) occur in patients with systemic lupus erythematosus (SLE) and in several strains of mice that spontaneously develop a lupus-like illness. An increase in circulating IC might occur as a result of increased IC formation or decreased IC clearance. Previous work with one murine lupus strain, NZB/W, demonstrated normal clearance of soluble IC. We studied the in vivo behavior of stable model IgG IC in two murine lupus strains. MRL/l and BXSB, and in two normal strains, C3H/HeN and BALB/c. IC were injected intravenously, and blood radioactivity was measured over 3 hr. A clearance curve was derived for each mouse using the Marquardt-Levenberg curve-fitting method. A formula is derived which predicts the formation rate of IC in each animal from the blood level of IC and the clearance curve parameters. All mice exhibited biphasic exponential clearance of IC over 3 hr. Clearance of IC was significantly slower in MRL/l mice than in normal strains (P less than 0.02), and clearance in BXSB mice was significantly faster than in normals (P less than 0.02). However, the derived formula suggests that the observed differences in IC clearance have only a small effect on the blood levels of IC in the mice studied, and therefore suggests that the major factor which accounts for increased blood levels of IC in lupus mice is an increase in the IC formation rate.     

Macrophage interactions with antibodies and soluble immune complexes

In vitro studies aimed at characterising (1) the binding of monomeric immunoglobulins from a variety of animal species to homologous mononuclear phagocytes, (2) the enhancement in phagocyte binding when antibodies are reacted with soluble antigens to form complexes of defined size, (3) the kinetics of complex ingestion and catabolism by macrophages and the biochemical mechanisms involved, (4) the role of complement in soluble complex catabolism and (5) the stimulatory effects of soluble complexes on phagocyte activity are reviewed. Insights gained from these studies into the in vivo clearance of soluble complexes and into the part played by circulating immune complexes in disease are discussed.     

B lymphocytes in lymph nodes and peripheral blood are important for binding immune complexes containing HIV-1

We investigated the interaction of HIV immune complexes (HIV IC) with mononuclear cells from lymph nodes and blood. While antibody alone did not affect binding of HIV IC to mononuclear cells, antibody plus complement increased binding by as much as 10-fold and complement alone also increased binding slightly. Most of the increased binding of HIV IC to mononuclear cells was blocked by heat-inactivation of complement and by OKB7 monoclonal antibody, indicating that virus binding was to CR2 on B cells. A similar pattern of antibody and complement dependence for binding of HIV IC was observed with two model systems; Raji and Arent B-cell lines. Most of the HIV IC that bound to lymph node cells were not internalized, but remained on the cell surface and were gradually released. However, even after 48 hr some HIV IC could be detected bound to cells. Under certain conditions, HIV IC were infectious for T cells if bound to B cells but not infectious if added directly to T cells. Additionally, HIV IC bound to B cells led to higher virus replication. These studies show that B lymphocytes from blood and lymph nodes can transfer infectious HIV IC to T cells.     

Limitations of quantitative photoacoustic measurements of blood oxygenation in small vessels

We investigate the feasibility of obtaining accurate quantitative information, such as local blood oxygenation level (sO2), with a spatial resolution of about 50 microm from spectral photoacoustic (PA) measurements. The optical wavelength dependence of the peak values of the PA signals is utilized to obtain the local blood oxygenation level. In our in vitro experimental models, the PA signal amplitude is found to be linearly proportional to the blood optical absorption coefficient when using ultrasonic transducers with central frequencies high enough such that the ultrasonic wavelengths are shorter than the light penetration depth into the blood vessels. For an optical wavelength in the 578-596 nm region, with a transducer central frequency that is above 25 MHz, the sensitivity and accuracy of sO2 inversion is shown to be better than 4%. The effect of the transducer focal position on the accuracy of quantifying blood oxygenation is found to be negligible. In vivo oxygenation measurements of rat skin microvasculature yield results consistent with those from in vitro studies, although factors specific to in vivo measurements, such as the spectral dependence of tissue optical attenuation, dramatically affect the accuracy of sO2 quantification in vivo.     

In vitro uptake and digestion of immune complexes containing guinea-pig IgG1 and IgG2 antibodies by macrophages.

The uptake and digestion of immune complexes by peritoneal macrophages from oil-stimulated guinea-pigs were studied using 125I-labelled guinea-pig IgG1 and IgG2 antibodies to hen ovalbumin. When the IgG1 or IgG2 antibody was incubated with homologous antigen at antigen-antibody ratios ranging from 0-01 to 10, the complexes produced were preferentially taken up by macrophages in the absence of complement and digestion by intracellular enzymes. The uptake and digestion of complexes reached a maximum at an antigen-antibody ratio of 0-1-0-5. In this respect, the IgG1 antibody was indistinguishable from the IgG2 antibody having a cytophilic activity. These observations suggest that some conformational changes in both the IgG1 and IgG2 antibodies or specific molecular arrangement in the lattice work of both the complexes may increase the strength of binding of complexes to macrophages, independently of the difference in biological activities between these two antibodies.     

Characterization of interactions involving anti-metatype antibodies and immune complexes.

Immunizations of high affinity anti-fluorescein monoclonal antibody 4-4-20 affinity labeled with fluorescein 5-isothiocyanate into a rabbit elicited antibodies specific for the liganded conformation of 4-4-20 (termed "anti-metatype" antibodies). Reaction of liganded 4-4-20 with anti-metatype antibodies caused significant delay (up to 23-fold) in the rate of dissociation of fluorescein ligand from the active site. In this study, structural analogues of fluorescein, including fluorescein 5-isothiocyanate, fluorescein 6-isothiocyanate, 5-dichlorotriazinyl aminofluorescein and 5-carboxyfluorescein, were bound by monoclonal antibody 4-4-20 and anti-metatype antibody reactivity was observed through delay in the dissociation rate of ligand from Mab 4-4-20. Significant delays (ranging from 5- to 242-fold) were observed for all structural analogues examined indicating that 4-4-20 maintained similar but not necessarily identical conformations upon binding fluorescein structural analogues. Additionally, fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate were conjugated to carrier molecules of increasing mol. wt (ranging from 225 to 14,600 D) in an attempt to sterically interfere with "metatopes" at the mouth of the active site and localize regions of anti-metatype antibody binding. These fluorescein-conjugated compounds were reacted with 4-4-20, and binding of anti-metatype antibodies delayed dissociation rates from 24- to greater than 1500-fold. These results indicated that the mechanism whereby anti-metatype antibodies delay the release of fluorescyl ligands from the active site probably does not solely involve steric hindrance of the ligand due to binding of anti-metatype antibodies at the mouth of the active site. Studies with 4-4-20 Fab fragments and a single chain derivative of 4-4-20 (consisting of the variable regions tethered by a 14 amino acid linker) indicated that anti-metatype reactivity was specific for the immunoglobulin variable region.