Preparative Isolation of Immune Complexes from Serum by Sucrose Gradient Ultracentrifugation

Immune complexes were isolated by ultracentrifugation in sucrose gradients (20-65% (w/w]. the centrifugation procedure was demonstrated to be isopycnic. The banding density of the complexes was influenced by the chemical nature and molecular size of the antigen and by the antigen to antibody ratio. The method was applied for preparative isolation of immune complexes from patients with systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and uncomplicated psoriasis.     

Isolation of Model Soluble Immune Complexes in the Fluid Phase by Monoclonal Anti-IgG- Ferritin Antibodies

Ferritin conjugates of monoclonal IgG anti-human gamma chain (anti-IgG-F) were reacted with soluble heat aggregates of IgG (A-IgG) and with soluble DNA-anti-DNA complexes to increase the S rate of the model soluble immune complexes (ICx) and thus facilitate isolation of ICx in the fluid phase and provide an immunochemical marker for subsequent ultrastructural analysis. A-IgG appeared as globular or curvilinear structures with individual IgG molecules arranged in a random fashion. The technique appears promising for characterization of other soluble ICx.     

Neutrophil Chemotaxis Induced by Immune Complexes

In the current work we have analyzed the ability of different soluble immune complexes (IC) prepared with IgG antibodies to induce neutrophil chemotactic responses in vitro. While, in all cases, IC were able to induce neutrophil migration in a concentration-dependent fashion, IgG antibodies alone were completely unable to induce locomotor responses. Checkerboard analysis indicated the chemotactic nature of motility. On the other hand, chemotaxis induced by IC was markedly inhibited by IV.3, a monoclonal antibody (mAb) to FcγRII, slightly reduced by 3G8 F(ab′)2, a mAb to FcγRIII, and nearly abrogated by both mAbs. The impact of IC on neutrophil migration induced by FMLP was also studied. We found that when a suboptimal concentration of FMLP was employed, the simultaneous addition of IC increased the migration acting in additive form. The significance of these results is discussed.     

类风湿关节炎患者血清IgG-IgM型类风湿因子免疫复合物检测的临床意义

目的 探讨血清IgM型类风湿因子与变性IgG抗原形成的复合物定量检测对类风湿关节炎诊断的临床意义.方法 收集类风湿关节炎(RA)患者36例、非RA患者41例和体检健康者40名作为研究对象.用包被有鼠抗人μ链抗体的ELISA孔板及HRP标记的兔抗人IgG对血清中IgM型类风湿因子(RF)与变性IgG抗原形成的免疫复合物水平进行检测,同时用胶乳凝集法检测RF,并对二者结果进行相关性分析;用ELISA试剂盒进行抗环瓜氨酸肽抗体(抗CCP抗体)的定性检测,并比较其与IgG-IgM型RF免疫复合物对于RA诊断的敏感性和特异性.结果 待检血清最佳稀释倍数为100倍.IgG-IgM型RF免疫复合物对于RA的敏感性为72.2％,特异性为95.3％.IgG-IgM型RF免疫复合物OD值与RF阳性程度相关系数为0.687(P ＜0.01).结论 血清IgG-IgM型RF免疫复合物水平对RA具有较高的敏感性和特异性,可作为RA诊断的参考指标.     

Human IgG Rheumatoid Factors and RF-Like Immune Complexes Induce IgGl Rheumatoid Factor Production in Mice

The synovial fluid of patients with rheumatoid arthritis (RA) was found to contain IgG and /or IgG-containing immune complexes (ICs) that stimulated an intense antibody formation when injected into mice of certain strains, notably of NZ background. The response was characterized by high and sustained levels of IgG1 antibodies with rheumatoid factor (RF) activity. In the study described, we investigated whether it is the antibodies with RF activity in the synovial fluid, that are responsible for stimulation of mouse RF in vivo. Different mouse strains were injected with synovial fluid from a seropositive RA patient (RA-SF), with human monoclonal antibodies with RF activity, with a human non-RF monoclonal antibody or with different preformed RF-like antibody antibody (Ab-Ab) ICs. The experimental mice were monitored subsequently for IgGl RF production. IgGl RF antibodies were found in all strains (NZB, BALB/c and CBA) injected with Ab-Ab ICs formed at equivalence, but only in NZB using RA-SF or human monoclonal antibodies with RF activity. Optimal production of IgG I RF by Ab-Ab ICs required the integrity of Fc and F(ab)'₂ portions respectively of the antibodies; soluble and truncated ICs were less effective. Further studies demonstrated that the IgGl RF response was not simply the result of a specific immune response against human IgG, since humoral immunity against human IgG was induced only when combined with an efficient adjuvant. During a typical adjuvant-associated primary response specific antibodies of IgM, IgG1 and IgG2a isotypes were found, i. e. quite different from the selective IgGl response induced by RF-like containing immune complexes. This conclusion is substantiated further by the clear differences in responses to IgG containing fraction obtained from RA-SF in NZ mice compared to other strains. Our findings argue for a different type of reaction leading to the selective IgGl response and might aid in elucidating the mechanisms for chronic production of antibodies with RF activity in patients with RA.     

Demonstration of Circulating IgG-Lactate Dehydrogenase Immune Complexes by Crossed Immunoelectrophoresis

Modified crossed immunoelectrophoresis was used to demonstrate complexes between IgG and lactate dehydrogenase in a human serum. The complexes were probably antigen-antibody complexes, since the Fab fragments, but not the Fc fragments, of the IgG molecules combined with normal human lactate dehydrogenase. The IgG autoantibodies reacted with both types of poly-peptide chains present in lactate dehydrogenase. The antibodies did not lower the catalytic activity of any of the 5 isoenzymes normally occurring in human serum. The antibodies formed exclusively soluble complexes with the isoenzymes and reacted not only with human lactate dehydrogenase, but also with that of rabbit and cow.     

Phagocytosis of IgA Immune Complexes by Human U937 Cells

Human promonocytic cell line U937 can express both IgAFc receptors ( FcαRⅠ, CD89 ) and IgG Fc receptors(FcαγⅠ, FcγRⅡ and FcγRⅢ)[1]. These receptors canmediate a variety of cell reactions including phagocytosis ofimmune complexes ( ICs ), degranulation, respiratorybursts, release of cytokines and enhancement of antibody dependent cell mediated cytotoxicity (ADCC) [2]. AfterIgA and IgG form ICs with their corresponding antigens,the ICs are bound by FcR on phagocyt…     

Neutrophil Cytotoxicity Induced by Immune Complexes prepared with Cationized Antibodies

Here we analyse the ability of soluble imtnune complexes (IC) prepared with cationized antibodies to induce cytotoxic responses mediated by neutrophils. While eationized IC induced high levels of cytotoxicity, control IC induced very low levels of response. Inhibition of cytotoxicity by catalase but not by three haemenzyme inhibitors suggests that oxygen-dependent but myeloperoxidase-independent mechanisms are responsible for cytolysis. While the response induced by control IC was enhanced by cytochalasin B and was not modified by colchicine, that induced by cationized IC was markedly inhibited by cytochalasin B and significantly enhanced by colchicine. Cytotoxicity induced by cationized IC was completely abrogated by monoclonal antibodies to FcγRII. Using control IC, a partial inhibition was observed employing either anti-FcγRII or anti-FcγRIII monoclonal antibodies. Treatment of neutrophils with ehemotrypsine or pronase significantly enhanced cytotoxicity induced by cationized IC but not by control IC. We also found that non-specific absorptive mechanisms appear to play an important role in the binding of cationized IC, but not control IC, to the neutrophil surface. The significance of these results is discussed.     

Circulating Immune Complexes in Systemic Scleroderma

Circulating immune complexes were detected by the immunoelectrophoretic method in 18 of 29 (62 per cent) of patients with systemic scleroderma. The presence of immune complexes did not correlate with that of antinuclear antibodies to dsDNA, DNP, RNP, and Sm. The mean levels of immunoglobulins G, A, and M as well as of C3 were significantly higher in patients with systemic scleroderma than in blood donors.     

Circulating Immune Complexes in Hashimoto's Thyroiditis

Circulating immune complexes (IC) were determined in sera from 41 patients with Hashimoto's thyroiditis by a polyclonal rheumatoid factor (pRF) assay based on the inhibition of the agglutination of IgG-coated latex particles. Elevated levels of IC were found in 63% (26/41) of the sera. There was a significant correlation (Rho = 0.91, P < 0.001) between results obtained before and after treatment of sera with dithiothreitol (DTT). By precipitation with 2.5% polyethylene glycol (PEG) before pRF inhibition assay, the activity of IC was found in only 7% (3/41) of the sera. Size chromatography studies of the sera showed the inhibitory activity predominantly in the intermediary region. When found in the IgM-region the activity was not reduced by DTT. By use of a polyethylene glycol complement consumption test (PEG-CC) the occurrence of IC was 10% (4/41). It was not possible to find any correlation between the detectable IC and the presence of microsomal, thyroglobulin, or thyroid-stimulating antibodies. Based on our studies the sizes of IC seemed to be heterogeneously distributed and the majority were not precipitated by PEG (2.5%, final concentration). The antibodies involved in the formation of complexes seemed to be of IgG or IgA classes. HLA-D typing of the patients showed a non-significant association between HLA-Dw5 and low levels of IC while the presence of HLA-Dw4 was significantly associated with a high level of IC (P < 0.05).     

Circulating Immune Complexes in Systemic Scleroderma

Circulating immune complexes were detected by the immunoelectrophoretic method in 18 of 29 (62 per cent) of patients with systemic scleroderma. The presence of immune complexes did not correlate with that of antinuclear antibodies to dsDNA, DNP, RNP, and Sm. The mean levels of immunoglobulins G, A, and M as well as of C3 were significantly higher in patients with systemic scleroderma than in blood donors.     

The Detection of Antigens in Immune Complexes

A procedure is described for the purification of soluble immune complexes (IC) from biologica fluids and for the detection of antibody-bound antigen. A model IC, prepared with various amounts of human serum albumin (HSA) and constant amounts of anti-HSA, gave a 44%, recovery after gel filtration on Sephacryl S-300, affinity chromatography on protein A-Sepharose, and concentration of eluates therefrom before analysis by sodium dodecyl sulphate-polyacryl-amide gel electrophoresis. It was calculated that 20 ng of antibody-bound HSA should be detectable when polyacrylamide gets are subjected to a direct radioimmunoassay involving of the use of rabbit anti-HSA. ¹²⁵I-protein A. and autoradiography. Thus defining the sensitivity of the procedure for detecting IC-bound antigen in a model system. The procedure has direct relevance to the examination of IC of unknown composition present in sera of individuals with various diseases.     

Retention of Immune Complexes by Fc Receptors on Mouse Follicular Dendritic Cells

Follicular dendritic cells (FDC) are located inside lymph follicles and are mainly characterized by their capacity to retain antigens. We investigated this aspect in mice lymph nodes by using bovine serum albumin (BSA) labelled with 5-nm colloidal gold particles and homologous anti-BSA antibodies bound to 20-nm gold particles. Gold-labelled BSA injected alone in non-immunized mice was only rarely found in FDC cytoplasmic interdigitations. Injected in the form of immune complexes, it was retained by FDC. Antigen-free anti-BSA antibodies injected under similar conditions as immune complexes were always found in draining lymph nodes in the same locations as BSA-anti-BSA immune complexes. F(ab')2 from mouse immunoglobulins linked to colloidal gold particles were very rarely found between the FDC extensions, whereas it was intensely phagocytosed by macrophages. Our study permitted precise ultrastructural localization between FDC cytoplasmic extensions or inside macrophages and other cells of the lymph nodes, but it also pointed out that homologous antibodies linked to colloidal gold particles might be retained by FDC in the absence of antigens. These observations, carried out with colloidal gold, were checked by using 125I-labelled anti-BSA antibodies. Complement activation determinations of gold-labelled antibodies or immune complexes showed that antibodies or immune complexes fixed on colloidal gold particles do not activate the complement. This observation enabled us to conclude that Fc receptors play a significant part in the retention of gold-labelled antibodies or immune complexes by FDC of lymph nodes.     

Association of Endothelin with Lung Hemorrhage Induced by Immune Complexes in Rats

The participation of endothelins (ETs) in a model of neutrophil-dependent lung injury induced by intrabronchial instillation of rabbit antibodies to ovalbumin followed by i.v. injection of the antigens (Arthus reaction) was investigated. Hemorrhagic lesions were evaluated by measuring the extravasations of hemoglobin in lung parenchyma. From 5 min to 24 h after the Arthus reaction (AR), endothelin (ir-ET) levels in bronchoalveolar lavage fluid (BALF) and in plasma were measured by radioimmunoassay. BALF levels of ir-ET were not different between control and AR animals for the first 90 min after the antigen challenge but increased from 2 to 24 h after induction of AR. ET levels in the plasma did not change from the respective controls over the same 24 h period. Increased ir-ET in BALF was not affected by pretreatment with L-NAME (30 mg/kg, i.v.). A PAF antagonist (BN52021; 5 and 10 mg/kg, i.v.) increased ET content in BALF and decreased the intensity of the AR. Thiorphan (2 mg/kg, i.v.) inhibited the AR-induced hemorrhagic lesions in lungs. An ET_A receptor antagonist, BQ-123 (1 mg/kg, i.v.) potentiated, whereas the ET_B antagonist, BQ-788 (1 mg/kg, i.v.) inhibited the lung hemorrhage. It is concluded that ETs are released during and play a role in the lung AR.     

Dissociation of Soluble Antigen—Antibody Complexes by Rheumatoid Factor IgM

Intact rheumatoid factor (RF)-active IgM dissociated soluble antigen-antibody complexes formed in the antigen excess zone, whereas trypsin-digested protein had less effect. The dissociation mechanism involved an interaction between the RF IgM and the Feγ of antibodies in the complexes. RF-active IgM had no demonstrable effect on antigen—antibody complexes when the antigen or the antibody had been immobilized. This was true irrespective of whether the experiments were performed in the antigen or in the antibody excess zone and despite binding between RF IgM and the immobilized proteins.     

Immune Complexes in Human Melanoma: A Consequence of Deranged Immune Regulation

Circulating immune complexes were detected in 62 individuals with malignant melanoma by precipitation with isolated human C1q and polyclonal rheumatoid factors. In 56 patients the C1q deviation assay showed low to moderate levels of complexes, with increased amounts with advancing stage of disease. Both heavy (>19S) and intermediate (7S to 19S) varieties were present, and complexes containing tumor antigen-antibody or antibody- anti-antibody were identified. Complexes were found in the kidneys of one patient with malignancy and the nephrotic syndrome and in two further patients with melanoma in whom there were DO clinical manifestations of nephrosis. Serial determinations in 51 patients showed slow cyclic variations in the levels of complexes and fluctuations in response to therapy. The coexistence of anti-antibodies, immune complex disease, and anergy in melanoma patients may indicate a deranged immune regulation consequent to chronic antigenic stimulation by the tumor.     

The Isolation of an Antigen Binding Factor from Cultured Murine T-Lymphocytes

BALB/c mice were immunized with the random sequence polypeptide [Glu⁶⁰ Ala⁴⁰] (GA). T-lymphocyte suspensions were prepared from lymph nodes, radiolabelled with ¹²⁵I and cultured for 18 hours. Supernatant, fluids were collected and passed over an immunoadsorbant composed of [D-Glu⁶⁰ D-Ala⁴⁰] (D-GA) bound to Sepharose. The non-adherent fraction was then applied to GA Sepharose and the adherent material eluted with 2 M NaSCN. The eluate was relabelled with ¹²⁵I, readsorbed to GA Sepharose and oluted. This fraction bound well to GA-Sepharose (50-80%) but not to the enantiamorphic D-GA Sepharose (5-10%). This material did not react with goat antisera directed against murine IgG, IgM or IgA. It was capable of binding to rabbit antiidiotypc sera raised against BALB/c anti-GA, but not to those antibodies raised against BALB/c anti-GAT. It also showed some reaction with rabbit antiserum to a rat T cell factor (TCF). Polyacrylamide gel electrophoresis revealed a major component with an approximate molecular weight of 63,000 which was not affected by reduction and alkylation.