Aggregated Human Gamma-globulin Research Articles

Possible role of free radical altered IgG in the etiopathogenesis of rheumatoid arthritis.

Alteration of IgG by oxygen-derived free radicals has been implicated in an in vivo process which renders IgG autoantigenic and leads to the production of rheumatoid factor (RF) and the perpetuation of inflammation, as in rheumatoid arthritis (RA). In this study the impact of UV irradiation on IgG was investigated as well as the ability of RF to bind to UV-altered gamma globulin. Inhibition studies of the binding of 125I aggregated human gamma globulin (AHG) to RF-coated sepharose beads show that UV-irradiated IgG is able to bind RF to the same extent as AHG. Binding studies to 125I-C1q proved that UV-irradiated IgG could bind the first complement component, but also that the complement system could be activated as illustrated by the C3a generation. These results support the hypothesis that free radical damage to gamma globulins plays a role in the chronicity of the inflammatory reaction in RA.

A novel bioassay to predict the clinical response to cryofiltration treatment. A preliminary report.

Phagocytosis by normal human mononuclear monocytes grown in vitro was examined after incubation with known concentrations of aggregated human gamma-globulin (AHG) and in plasma obtained from patients suffering from immunologic diseases. When the phagocytes were pretreated with AHG, an inverse relationship between amounts of AHG and phagocytosis of radiolabeled Candida albicans was found. Inhibition of phagocytosis by patient plasma was independent of other laboratory determinations including antinuclear antibody, C3, C4, CH50, DNA-binding and circulating immune complexes. Plasma from 5 patients subjected to cryofiltration treatment (CFT) demonstrated less inhibition after than before CFT. This lessened inhibition of phagocytosis in vitro correlated with the clinical response due to CFT. The response to CFT could not be predicted by the conventional laboratory parameters investigated. Improvement between pre- and post-CFT plasma estimated by using the present bioassay may provide a quantitative description of which patient may benefit from plasmapheresis treatment procedures.

Active transformation of tolerogenic to immunogenic signals in T and B cells by 8-bromoguanosine.

The injection of deaggregated human gamma-globulin (DHGG) into A/J mice results in the establishment of a state of unresponsiveness to subsequent challenge with immunogenic aggregated human gamma-globulin (AHGG). Administration of the B cell activator 8-bromoguanosine (8BrGuo) 3 hr after administration of DHGG converts the tolerogen to an immunogen and results in an antibody response of even greater magnitude than the primary response elicited by AHGG alone. Adoptive transfer studies with separated populations of T and B cells demonstrated that although transformation of the tolerogenic signal to an immunogenic signal involves effects of 8BrGuo on both T cells and B cells, the major effect appears to be activation of antigen-specific T cells that would otherwise become tolerant. Modulation of T cell tolerance could conceivably be mediated either by direct or indirect mechanisms. Interestingly, optimal responsiveness of B cells from animals treated with DHGG and 8BrGuo is not a T cell-independent event, but requires antigen-reactive T cells. 8BrGuo is not able to override unresponsiveness when given 10 to 20 days after tolerance induction, at a time point when both T and B lymphocytes are tolerant. However, when given at day 60, when T cells (but not B cells) remain tolerant to this antigen, the nucleoside is able to terminate the tolerant state prematurely, possibly by providing an alternate T helper-like signal directly to B cells or by recruiting nonspecific functional T helper cells.

The origin of follicular dendritic cells in the mouse and the mechanism of trapping of immune complexes on them.

F1----parental bone marrow chimeras between CBA and B10 mice were used to show that even after 1 year follicular dendritic cells (FDC), also termed dendritic reticular cells, are not derived from bone marrow stem cells. However clusters of cells wrapped in the dendritic processes of FDC, isolated by enzyme digestion of the spleen contain B cells originating from donor marrow. This implies that evidence for the presence of antigens such as Ia-like detected by others (Gerdes, J. and Stein, H., Clin. Exp. Immunol. 1982. 48: 348) by immunohistology in or on FDC clusters should be interpreted with caution. Deposition of immune complexes on mouse spleen FDC depends upon the presence of a radio- and cyclophosphamide-sensitive population of cells, although FDC themselves are resistant to such treatments. After whole body irradiation the capacity to localize fluorescein isothiocyanate-labeled aggregated human gamma globulin can be restored by normal or T-deprived spleen cells, but restoration requires an interval of more than 1 though less than 5 days. These findings are compatible with the evidence of Gray et al., Eur. J. Immunol. 1984. 14: 47, that in the rat immune complexes are transported to FDC by a sessile population of marginal zone lymphocytes, as first suggested by Brown et al., Immunology 1973. 24: 955.

Effect of Fc receptor blocking on human natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells.

The effect of Fc receptor (FcR) blocking by aggregated human gamma-globulin (AGG) was studied on natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of [3H]TdR-prelabelled HEp-2 cells. LDCC was evaluated in a 24 h assay at 50:1 effector-target cell ratio in the presence of 25 micrograms/ml concanavalin A (Con A). Under these conditions but without Con A considerable NCMC was not elicited by normal lymphocytes. FcR blocking by AGG treatment of effector cells resulted in a significant NCMC activity to HEp-2 targets. In contrast, AGG treatment profoundly depressed LDCC. Monocyte depletion of effector cells had no major influence on the effect of AGG on NCMC and LDCC activities. An interference of FcR blocking by AGG and LDCC in response to Con A is suggested.

Critical aspects of immune complex assays employing polyethylene glycol

Treatment of artificial immune complexes (ICs) with 2.5% polyethylene glycol (PEG) — conditions under which C1q-binding activity is routinely measured in the fluid phase — produced marked changes in molecular size as determined by Sepharose 6B chromatography. The effect of PEG on the C1q-binding capacity of ICs, was therefore investigated using a solid phase (SP) system. PEG enhanced the binding of aggregated human gammaglobulin (AHG) and artificial ICs to SP-C1q and, in reverse experiments, also increased the binding of C1q to SP-AHG. The degree of enhancement varied according to the Ag: Ab ratio employed; the binding of ICs formed in moderate Ab excess was only modestly enhanced but that of complexes formed at slight Ab excess, equivalence and Ag excess was markedly elevated. The profile of PEG-induced enhancement of binding paralleled that of similar ICs in the C1q fluid phase system, suggesting that C1q binding in the latter may be influenced by PEG. However, the C1q-binding activity of in vivo-formed ICs seemed to be relatively unaffected by PEG since enhanced binding was comparable in control and pathological sera. The results indicate that PEG causes cross-linking and aggregation of ICs (and possibly other serum proteins) which may alter their biological activity and hence influence the results of IC assays that employ this agent.

Measurement of circulating immune complexes in human sera by enzyme immunoassay.

Two methods, based on enzyme immunoassay, are described for the detection and quantitation of circulating immune complexes (CIC) in human sera. Aggregated human gamma globulin (AHG) or immune complexes in human sera are bound to complement receptors on Raji cells or to Clq adsorbed on to the plastic surface of microtitre plates. The bound complexes are subsequently detected using peroxidase-conjugated anti-human immunoglobulin antisera. The assays offer a benefit over previously described assays in that they use cheap, commercially available antisera and enable the detection of immune complexes composed of varying immunoglobulin classes.

An alternative decomplementation method for the measurement of the survival of human erythrocytes transfused into rats.

An alternative technique for measuring the survival of51Cr-labelled human erythrocytes transfused into rats is described, in which aggregated human gamma-globulin is substituted for cobra venom factor as a decomplementing agent.

Detection of immune complexes using a solid-phase C1q polystyrene ball assay

A polystyrene ball C1q solid-phase assay (PSB C1q SPA) has been developed for quantitating immune complexes in human serum. While similar to the previously reported solid-phase C1q assays in principle, the use of polystyrene balls with a specular finish has resulted in assay with significantly improved accuracy, sensitivity and reproducibility. The sensitivity of the assay based on the amount of AHGG bound per μg C1q added was approximately 12-fold higher in the PSB assay compared to the polystyrene tube solid-phase C1q method. In order to correct for variable background contributions in different samples, values obtained with heat-inactivated C1q were subtracted from each experimetal result. Reproducibility studies yielded a coefficient of variation (CV) of 10 and 4% in day-to-day assays using 25 μg and 100 μg AHGG/ml normal serum, respectively compared to 11–15% for the tube technique. Within run measurements gave a CV of 37 and 20% at the low and high levels of AHGG. Aggregated human gamma-globulin (AHGG) was used as a model immune complex and when chromatographed on Biogel A-15m yielded major fractions at ⩾15,000,000 and 150,000 daltons. Maximum binding of AHGG to PSB C1q occurred with aggregates greater than 15,000,000 daltons. Optimum binding of human albumin-anti-albumin complexes in the PSB C1q SPA occurred at a molar ratio of 1:1.5. The size distribution of this complex active in the assay determined by sucrose density gradients was 14–32 S with peaks at 21 and 27 S. A normal range of immune complexes was determined as 15±8 μg AHGG equivalents (±2 S.D., n = 65). Approximately 70% of rheumatoid arthritis, linear scleroderma, vasculitis, Sjögren's and glomerulonephritis and 40% of SLE patients were above 2 S.D. of normal. SLE patients demonstrated elevations in immune complexes only during periods of increased disease severity. The assay was a useful monitor of plasmapheresis in an SLE patient, showing decreases in immune complexes after each plasmapheresis. DNA did not interfere with AHGG binding whereas lipemia prevented detection of added AHGG.

K cell activity of murine-cultured lymphocytic leukemic cell line independent of other Fc receptor functions.

A murine-cultured lymphocytic leukemic cell line, SC-1, showed remarkable cytotoxicity against 51Cr-labeled rabbit erythrocytes as target cells in the presence of antitarget antibodies in vitro. This cytotoxicity was complement independent, inhibited by the addition of aggregated human gamma-globulin, and reduced by pepsin digestion of antitarget IgG. Moreover, SC-1 cells also induced cytotoxicity to trinitrophenyl-modified rabbit erythrocytes in the presence of antitrinitrophenyl antibodies but not unmodified rabbit erythrocytes used as bystander cells. SC-1 cells which showed such K cell activity, however escaped from Fc receptor detection by erythrocyte antibody rosettes and FITC-conjugated aggregated human gamma-globulin binding. In morphological studies using the scanning electron microscope, SC-1 cells to induce cytotoxicity through a light attachment to target cells via 'point contact' of the microvilli. From these findings the characteristics of Fc receptors on effector cells in the mechanism of antibody-dependent cellular cytotoxicity was discussed.

Inhibition of polymorphonuclear leukocyte chemiluminescence for detection of immune complexes in human sera.

An assay for the detection and quantitation of immune complexes is described. Experimental immune complexes or aggregated human gamma globulin (AHG) were incubated with polymorphonuclear leukocytes (PMN). After challenge of the PMN with opsonized zymosan, chemiluminescence was recorded in a scintillation spectrometer. A quantitative inhibition of chemiluminescence could be demonstrated by the interaction of PMN with immune complexes or AHG. Experimental immune complexes of bovine serum albumin-anti-bovine serum albumin were formed and tested by this assay, and immune complexes formed near antigen excess were best described by this technique. The technique was used to demonstrate immune complexes in the sera from patients with systemic lupus erythematosus, rheumatoid arthritis, and vasculitis. Immune complexes were quantitated by reference to a standard curve using AHG. By this technique, normal human sera had < 10 micrograms of AHG per milliliter of serum. Immune complexes at levels above this were detected in 9/15 patients with systemic lupus erythematosus, 18/30 patients with rheumatoid arthritis, and 2/5 patients with vasculitis. Therefore, this assay is a sensitive, simple method for measurement of circulating immune complexes in the sera of patients with certain connective tissue diseases.

Effect of d-penicillamine in vitro and in vivo on macrophage phagocytosis

Preincubation of rat peritoneal macrophages with d-penicillamine increased their uptake of labelled aggregated human gamma globulin ([ 125I]AHG) without affecting the rate of degradation of the aggregates. Administration of d-penicillamine (50 mg.kg −1. day −1 p.o.) to normal rats resulted in increased uptake of [ 25I]AHG by peritoneal macrophages after 4 days of treatment, but not after 14 and 28 days of treatment. In contrast, macrophages from rats with adjuvant arthritis treated with d-penicillamine (50 mg.kg −1. day −1 p.o.) exhibited an increased uptake of [ 125I]AHG throughout the course of the disease. Administration of d-penicillamine in vivo had no effect on the rate of degradation of [ 125I]AHG by freshly prepared macrophages. Culture for 24 hr in vitro prior to incubation with [ 125I]AHG led to a decrease in the rate of degradation of the labelled aggregates by macrophages from untreated or d-penicillamine-treated rats and from rats with adjuvant arthritis, but not by macrophages from d-penicillamine-treated adjuvant arthritic rats. It is suggested that d-penicillamine may exert a stimulatory effect on the reticuloendothelial function during chronic inflammatory disease, and that this effect may be mediated via an interaction with the macrophage plasma membrane.

Raji cell assay for immune complexes. Evidence for detection of Raji-directed immunoglobulin G antibody in sera from patients with systemic lupus erythematosus.

We asked whether binding of human immunoglobulin (Ig)G antibody reacting with Raji cells could be distinguished from binding of IgG immune complexes. Using a standard Raji assay employing 125I-IgG goat anti-human Fc gamma, we found that digestion of Raji cells with pronase reduced by 95% their ability to bind complement-fixed aggregated human gamma globulin and complement-fixed tetanus toxoid-antitetanus toxin complexes. However, binding at 37 degrees C of IgG from the sera of 16 patients with systemic lupus erythematosus (SLE) to pronase-digested Raji cells was reduced much less consistently and extensively (9-100% reduction; mean reduction of 51%). In more detailed studies of two SLE sera, sucrose density gradient centrifugation showed that greater than 50% of the IgG binding to undigested Raji cells sedimented in the 7S region. Pepsin digestion of immunoglobulin fractions from four SLE sera caused a reduction in SLE IgG binding to undigested Raji cells when detected with 125I anti-Fc gamma, but an increase when binding was detected with 125I-anti-Fab, suggesting that substantial SLE IgG can bind through F(ab')2 regions. Binding of IgG from SLE sera was not directed at neoantigenic sites induced by pronase digestion because binding activity was adsorbed with undigested cells as readily as with digested cells. Moreover, sera from 10 SLE patients that had negative Raji assays contained no IgG that bound to pronase-digested Raji cells. We conclude that much of the IgG bound at 37 degrees C to Raji cells from the sera of many patients with SLE does not represent immune complexes but is probably antibody directed toward sites on the Raji cell.

Induction and mode of action of suppressor cells generated against human gamma globulin. II. Effects of colchicine.

The ability of colchicine (Col) to interfere with suppressor cells specific for the soluble protein antigen human gamma globulin (HGG) has been examined. This interference may be the mechanism of the adjuvanticity promoted by Col. When injected into A/J mice at the appropriate time and concentration, both Col and cyclophosphamide promoted an adjuvant increase in the plaque-forming cell response to 100 micrograms of immunogenic, aggregated HGG. Col abrogated both the induction of suppressor cells when injected with 3 h of tolerization with deaggregated (DHGG) and the expression of previously induced suppressor cells when injected with the antigenic challenge. Interference with the generation and expression of antigen-specific suppressor cells had no detectable effects on the immunologic unresponsive state to HGG. Col did not interfere with the induction of tolerance at a dose (1 mg/kg) that abolished the generation of suppressor cells. Furthermore, the absence of colchicine-sensitive-suppressor cells during the establishment of tolerance had no observable effect on the duration of unresponsivness in either helper T- or B-lymphocyte populations. Finally, Col was not able to terminate the unresponsive state established by DHGG even when responsive splenic B cells could be demonstrated in tolerant animals. These data indicate that suppressor cells are not required for the establishment and maintenance of the unresponsive state to this antigen.

The Raji cell radioimmune assay for detecting immune complexes in human sera.

A sensitivie and simple procedure for the detection and quantitation of soluble complement (C)- fixing immune complexes in sera of patients with various disease states has been developed by utilizing C receptors on Raji cells. These cells lack membrane-bound immunoglobulin but have receptors for IgG Fc, C3b, C3d, and possibly with other C proteins. Uptake experiments showed that both aggregated human gamma globulin (AHG) and 7S IgG bound to receptors for IgG Fc; however, AHG reacted with C bound to cells only via receptors for C and this binding was much more efficient than via IgG Fc receptors. AHG was used as an in vitro model of human immune complexes and its uptake by Raji cells was quantitated by 125I-radiolabeled antihuman IgG. The limit of sensitivity of this test was 6 mug AHG/ml serum. The ability of Raji cells to detect AHG in serum depended on the amount of radioactive antibody used and the size of aggregates. The presence of an excess of C somewhat inhibited binding of AHG containing C to Raji cells. The efficient binding of AHG by receptors for C on Raji cells was used for the detection and quantitation of immune complexes in human sera. Raji cells were incubated with sera to be tested and then reacted with excess radiolabeled antihuman IgG; the amount of radioactivity bound to the washed cells was determined and referred to a standard curve of radioactive antibody uptake by cells previously incubated with increasing amounts of AHG in serum. Thereby immune complexes were detected and quantitated in serum hepatitis, systemic lupus erythematosus, vasculitis, subacute sclerosing panencephalitis, dengue hemorrhagic fever, and malignancies.

Connective tissue-degrading enzymes of human leukocytes.

Human leukocytes, when exposed to aggregated human gamma-globulin (AHGG) or immune complexes (isolated from RA synovial fluid) fixed to a cartilagenous surface, release neutral proteases that degrade the extracellular matrix of cartilage. The chondromucoprotein destruction by these proteases is suppressed by a variety of synovial fluids but is not susceptible to inhibition by trypsin, chymotrypsin, elastase inhibitors, or a combination of these agents. The inhibitory effect of synovial fluid can be reversed in the presence of increasing enzyme concentrations. Intact viable human polymorphonuclear leukocytes in the presence of AHGG also release a collagenase precursor that can be activated by limited proteolysis with trypsin or RA synovial fluids. Enzyme release (neutral proteases) by phagocytosing cells is inhibited by the antiinflammatory agents phenylbutazone and colchicine; these agents do not affect release of the collagenase precursor. However, the latent collagenase release is susceptible to inhibition when leukocytes are preincubated (prior to exposure to AHGG) with inhibitors of protein synthesis. Under these conditions, neutral protease release is unaffected.

RELEASE OF CARTILAGE MUCOPOLYSACCHARIDE-DEGRADING NEUTRAL PROTEASE FROM HUMAN LEUKOCYTES

The granule fraction of human leukocytes contains neutral protease capable of degrading the noncollagenous protein mucopolysaccharide matrix of cartilage at neutral pH in physiological salt solution. Cartilage degradation was monitored by quantitating the release of (35)S from labeled rabbit ear cartilage. Degradation of cartilage matrix occurs when intact viable human leukocytes are incubated with cartilage opsonized with aggregated human gamma globulin (AHGG). During a similar 4 h incubation period cells did not degrade uncoated cartilage or cartilage coated with nonaggregated gamma globulin. Cells remain viable during the enzyme release process as evidenced by the absence of a cytoplasmic enzyme marker (lactic dehydrogenase) in the supernatant and dye exclusion studies. The release of (35)S from labeled cartilage by human leukocytes in the presence of cartilage coated with AHGG (nonphagocytic enzyme release) was compared with the cartilage degrading activity of the supernatant from the same number of cells preincubated with a suspension of AHGG (phagocytic enzyme release). Nonphagocytic enzyme release by 5 x 10(6) cells provoked two to four times more (35)S and beta-glucuronidase (beta-G) release from cartilage than phagocytic enzyme release conditions. beta-glucuronidase was used as an indicator of the release of lysosomal granule enzymes. By the use of selected pharmacological agents it was possible to dissociate the enzyme release process from intrinsic enzyme (neutral protease) activity. Neutral protease and beta-G release by human cells in the presence of AHGG-coated cartilage was inhibited by 10(-5)M colchicine, whereas the protease activity, but not the release process, was inhibited by 10(-6)M gold thiomalate and 10% human serum. It is suggested that the release of a cartilage degrading neutral protease by viable human cells when exposed to AHGG might be a relevant model for the study of cartilage destruction as it occurs in rheumatoid arthritis.