Agg IgG Research Articles

P008 : AGGREGATED IgG CONTROL FOR PRONASE FC RECEPTOR (FcR) REMOVAL IN FLOW CROSSMATCH

Aim This study evaluated heat aggregated (Agg) IgG in IVIG and normal human serum (NRS) as a control for pronase FcR removal in Flow crossmatch. Pronase treatment of lymphocytes is used to remove FC receptors on B cells to reduce false B cell positive Flow crossmatches due to Agg IgG in patient serum. The effectiveness of pronase treatment can be compromised if there are too many cells or serum proteins present, a positive control is needed to verify that FcRs have been removed from B cells. Methods 1. To aggregate IgG, 5 ml of NRS or IVIG (Gammaguard 100 mg/ml) is heated at 63 °C for 20 min. 2. The working dilution for Agg IVIG is found by titering it on unpronased and pronased cells in a crossmatch. 3. A working dilution for Agg NRS is also found by titering it in a crossmatch, but first the Agg IgG is concentrated at 130,000g, 60 min at 4 °C. 4. The NRS Agg IgG pellet is resuspended in 1 ml PBS, pH 8.1 for titering. Results See the Fig. 1 , Fig. 2 . Conclusion This study demonstrates the ability to use heat Agg IgG in IVIG or from NRS as a control for pronase B cell FcR removal in Flow crossmatch.

Binding of polymeric IgG to fibronectin in extracellular matrices: an in vitro paradigm for immune-complex deposition

We have previously shown a biochemical interaction between fibronectin (Fn) and polymeric immunoglobulins (Igs), that we localized to the fourth and fifth N-terminal type I repeats ( 4 F1 · 5 F1 ) in Fn and the Fc portion of IgG. Therefore, we hypothesized that Fn, as a constituent of the extracellular matrix (ECM) may directly bind circulating immune complexes (ICs) causing their deposition, thereby contributing to the pathogenesis of IC diseases. As an in vitro paradigm to test this idea, we have generated Fn-containing ECMs from varied cells in culture and demonstrated a saturable dose-dependent binding of aggregated (agg) IgG, as a prototype of ICs, as well as the binding of both heat and cold aggregated purified type I cryoglobulins (CGs) to these ECMs. No binding was observed to ECMs (Matrigel) that do not contain Fn. Characteristic of our previous findings, polymeric but not monomeric IgG bound to the acellular Fn-containing ECMs. To further demonstrate the specificity of the interaction and implicate matrix Fn in the binding of aggIgG, complete inhibition of binding of aggIgG to Fn was achieved by blocking Fn on the ECMs with anti-Fn antiserum and by preincubation of the Ig aggregates with anti-human IgG antibodies. By competing the binding interaction with fluid phase Fn and the Ig-binding site on Fn, 4 F 1 · 5 F 1 , 70% inhibition was obtained. Additional experiments performed with purified CGs show that an identical dose-dependent increase in Fn binding occurred using both preformed and forming cryoprecipitates suggesting that Fn does not confer cryoprecipitation of CGs and that the specific association of Fn with cryoprecipitates probably results from their polymeric configuration. Our results support the notion that Fn, as it exits in expanding ECMs characteristic of glomerulonephropathies and rheumatoid synovial disease, specifically interacts with complexed/polymeric Igs, thereby perpetuating IC deposition and playing a role in the pathogenesis of IC diseases.

Effect of neuraminidase treatment on the expression of Fe IgG receptors on chicken embryonic bursa and thymus cells

Bursa and thymus cells from chicken embryos at different ages were analyzed for Fc IgG receptors by EA-rosette technique and by binding of heat aggregated chicken IgG (agg IgG) in the indirect immunofluorescence test. Neuraminidase treatment resulted in a substantial increase of agg IgG binding cells both in the embryonic bursa and thymus. The binding of agg IgG was shown to be specific for Fc IgG receptors, since IgM and F(ab′) 2 fragments were not bound to neuraminidase-treated embryonic cells. A far lower percentage of EA-rosette-forming cells than agg IgG binding cells were found both in untreated and neuraminidase-treated bursa and thymus cells. It was concluded that neuraminidase can reveal additional Fc IgG receptor sites mainly for agg IgG on embryonic thymus and bursa cells.

IgG Fc Receptor-bearing cells during early lymphoid cell development in the chicken

Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.

Studies on the chicken lymphocyte receptor for aggregated IGG

Chicken lymphocytes isolated from different lymphoid organs were analyzed for Fc IgG receptor by binding of heat aggregated IgG (agg IgG) in the indirect immunofluorescence test. The binding of agg IgG was shown to be specific for Fc IgG receptor, since IgM and F(ab′) 2 fractions were bound to the lymphocytes in a much less degree. The highest frequency of agg IgG binding cells, almost 100 %, was found in the bursa of Fabricius. In contrast to bursa, only a small percentage of thymus cells bound agg IgG. Dose-dependent experiments showed that binding of agg IgG to thymus cells takes place only at high concentrations of the aggregates whereas bursa cells bind agg IgG also at low concentrations. The finding indicates the existence of high and low avidity agg IgG receptors and/or different density of Fc receptors on bursa and thymus cells. A far lower percentage of EA rosette forming cells than of agg IgG binding cells in the various lymphoid organs may depend on the existence of two different Fc IgG receptors on the chicken lymphocytes.

Development of an immunochemical test to assess [formula omitted] inactivator function in human serum and its use for the diagnosis of hereditary angioedema

The antigenic and functional levels of C 1 inactivator were measured in 20 normal and pathological (hereditary angioedema) human sera. The average concentration of C 1 -In protein in normal human sera, determined by radial immunodiffusion, was 137 ± 11 μg/ml, while the level of C 1 -In functional activity, measured by hemolytic titration, was 128 ± 10 μg/ml. The HAE sera fell into two groups: HAE (group I)—sera containing low levels of C 1 -In antigen (average of 30% normal) and nondetectable functional C 1 -In; HAE (group II)—sera containing normal or high levels of C 1 -In antigen, but no detectable functional ( C 1 -In. These characterized sera were used to evaluate a simple immunodiffusion assay which was developed to replace the hemolytic C 1 -In functional test, which requires specialized reagents and is complex and thus impractical for many clinical laboratories. The assay consists of incubating each serum in the presence and absence of 2 mg/ml of the complement activator, aggregated IgG, for 60 min at 37°C, followed by determination of the residual C1r concentrations by radial immunodiffusion. During the incubation with agg IgG, C1r becomes activated and interacts with C 1 -In. This interaction masks the antigenicity of C1r which thus becomes undetectable in the serum. In NHS, the C 1r precipitin ring is totally lost after incubation with 2mg/ml agg IgG. On the other hand, sera lacking functional C 1 -In exhibit no change in the C 1r precipitin ring. Thus all sera lacking functional C 1 -In (HAE (group I) and HAE (group II)) were readily distinguished from those containing functional C 1 -In (NHS). The presence of nonfunctional C 1 -In protein did not interfere with the assay. This simple test which requires only IgG and anti-C1r, which are both commercially available, permits the routine screening of human sera for functional C 1 -In.

Active Disassembly of the First Complement Component, C1̄, by C1̄ Inactivator

Abstract In order to clarify its mechanism of disassembly, the first component of human complement, C1, was reconstituted from C1q, 125I-C1r, and 131I-C1s. This radiolabeled C1 bound tightly to aggregated IgG covalently linked to Sepharose 4B. The subsequent addition of C1̄ inactivator (C1̄-In) led to the rapid release of equimolar quantitites of 125I-C1r̅ and 131I-C1s̅ from the agg IgG. SDS-PAGE analyses of such eluates showed that one C1̄-In molecule was associated with each C1r̅ polypeptide chain and another C1̄-In molecule was bound to each C1s̅ polypeptide chain. The released C1r̅, C1s̅, and C1̄-In co-sedimented in sucrose density gradients with a rate of 9S and monospecific anti-C1s̅ pelleted all three proteins. Therefore, C1r̅, C1s̅, and C1̄-In, in a molar ratio of 1:1:2, respectively, are released from C1̄ as a complex. An analogous 9S complex containing C1r̅, C1s̅, and C1̄-In was generated in normal human serum after incubation with activators of the classical complement pathway. The C1r̅C1s̅(C1̄-In) complexes generated in both serum and the purified system were stable in the presence of EDTA. A diffusion coefficient of 2.3 × 10-7 cm2/sec was determined for the C1r̅C1s̅(C1̄-In) complex from its behavior on gel filtration. An m.w. of 330,000 was then calculated from its sedimentation and diffusion coefficients. This m.w. is consistent with the value of 382,000 for a molecule of composition C1r̅C1s̅(C1̄-In)2 obtained by summing the weights of the subunits. These results indicate that C1-In efficiently disassembles C1̄, thereby releasing two C1r̅C1s̅(C1̄-In)2 complexes per C1̄ molecule.

Binding of circulating immune complexes to human peripheral blood lymphocytes: Effect of complement

The mode of binding of immune complexes, with and without bound complement components, to normal human peripheral blood lymphocytes was examined. Heat-aggregated human IgG (agg IgG) was employed as an in vitro model of immune complexes. Immune complexes without bound complement or with bound C1, C4, or C2 inhibited antibody-dependent cellular cytotoxicity (ADCC) and EA rosette formation but not rosette formation with EAC. In contrast, immune complexes with bound C3b blocked EAC rosette formation but inhibited ADCC and EA rosette formation little if at all. These findings demonstrate that agg IgG, and thus immune complexes, without fixed complement or those with bound C1, C4, or C2, preferentially interact with the lymphocyte Fc receptor and that C3b-bound immune complexes bind to the lymphocyte surface C3b receptor.

Detection of immune complexes. The use of radioimmunoassays with Clq and monoclonal rheumatoid factor.

This study describes two sensitive, rapid, relatively simple, competitive inhibition radioimmunoassays for detecting immune complex. The tests are based on the inhibition of I125-Clq or I125-monoclonal rheumatoid factor (mRF) binding to an insoluble substrate, IgG-Sepharose. The assays can be performed in 5 h utilizing 10 micronl of serum. Heating of serum is not required and polyclonal rheumatoid factors do not interefere. With the two assays, a wide range of complexes of various size and complement fixing activity can be detected. The Clq test can detect complement fixing Ig complexes larger than 19S, while the mRF tests detect complexes of IgG as small as 8S irrespective of their complement fixing activity. Mouse, rabbit, and human aggregated IgG (agg IgG) can be detected in the Clq test, and human and rabbit agg IgG in the mRF test. As low as 4 microng/ml of isolated human agg IgG can be detected in the Clq test and 0.5 microng/ml in the rheumatoid factor test. Sensitivity is greater for mouse agg IgG. For pathologic sera which must be diluted to eliminate interfering factors, the sensitivity of the assay is approximately 10 times less. The Clq test showed marked inhibition by systemic lupus erythematosus sera with close correlation with CH50 levels and disease activity. The mRF test showed better correlation with rheumatoid arthritis sera. In addition, anionic macromolecules known to react with Clq and other Clq reactants that occur in pathologic sera such as the "low molecular weight" substances in systemic lupus erythematosus are also detected. These reactants are not detectable in the mRF test and can be eliminated in the Clq test by performing the test at higher ionic strength. The tests can be applied to the study of a variety of pathologic states where immune complexes appear to play a role.

Precipitation of 125I-labelled IgG aggregates by factors in sera of healthy individuals and of patients with rheumatoid arthritis.

Several factors in human serum are capable of precipitating soluble 125I-labelled heat aggregated IgG (agg IgG*). A study of the nature of these factors resulted in the development of two new methods for the detection and assay of anti-IgG-immunoglobulins (rheumatoid factors) in serum. One method detected rheumatoid factors of the IgG and the IgA classes which are capable of binding and coprecipitating with Clq and agg IgG* in an EDTA milieu. In a second method the serum was first heat inactivated and the assay was then made in a polymeric milieu where rheumatoid factors of the IgM as well as the IgG and IgA classes could be detected. 67 sera from patients with rheumatoid arthritis were tested with this method and rheumatoid factors were detected in all seropositive (as assayed with conventional rheumatoid factor tests) sera and in 58% of the seronegative sera. In the presence of certain anti-IgG-immunoglobulins or polymers, the precipitation of Clq and soluble agg IgG* is greatly enhanced, and we suggest that this can be used as a basis of a sensitive method for the assay of agg-IgG-binding activity of Clq.

Dissociation of phagocytosis, metabolic stimulation and lysosomal enzyme release in human leukocytes

In this paper studies are reported concerning the relationship between particle binding to the plasma membrane of human polymorphonuclear leukocytes (PMN's), phagocytosis, generation of oxidative metabolites, and the release of lysosomal enzymes by these cells. Superoxide (O2-) generation by, and lysosomal enzyme release from, normal PMN's and cytochalasin B-treated cells were measured. We have found that neither phagocytosis nor lysosomal degranulation are prerequisites for enhanced O2- generation. Cytochalasin B-treated PMN's, incapable of ingesting particles but still able to bind particles to membrane receptors, generated enhanced amounts of O2- when treated with serum-treated zymosan (STZ), a C3b receptor stimulus, or with aggregated IgG (agg IgG), an Fc receptor stimulus. Moreover, the soluble stimulators complement component C5a, phorbol myristate acetate (PMA), and calcium ions in the presence of the ionophore A23187, also increased the O2- production of these cells. In all cases a time and dose-dependent stimulation was found of both the O2- generation and the lysosomal enzyme release, but there was no correlation between ability of any stimulus to provoke enzyme release and its ability to stimulate O2- generation. When PMN's were preincubated with 5 X 10(-4) M hydrocortisone-Na-succinate, lysosomal enzyme exocytosis with the immune reactants was inhibited 16-35%. Hydrocortisone also inhibited O2- generation, except when STZ was used as the stimulus. Thus, in the case of stimulation of functional processes of PMN's via the C3b receptor, hydrocortisone inhibits membrane fusion without interfering with one of the early biochemical events (O2-production).

Immune complexes in sera and synovial fluids of patients with rheumatoid arthritis. Radioimmunoassay with monocylonal rheumatoid factor.

Evidence for the presence of immune complexes in blood, synovial fluid, and tisues of patients with rheumatoid arthritis (RA) includes low complement levels in blood and effusions, deposition of immunoreactants in tissues and vessel walls, precipitate formation after addition of monoclonal rheumatoid factor (mRF) to serum or synovial fluid. To quantitate immune complex-like material in RA patients, we developed a radioimmunoassay based on inhibition by test samples of the interaction of (125I)aggregated IgG (agg IgG) and mRF coupled to cellulose. This method could measure immune complexes of human antibody with hemocyanine prepared in vitro. The assay was not influenced by presence of polyclonal RF in test samples, nor by freezing and thawing. Normal levels of immune complex-like material in serum were less than 25 mug agg IgG EQ/ML. 12 of 51 RA sera examined (26%) contained more than 25 mug/ml. The presence of this material in RA sera was found to correlate with severity of disease, as measured by anatomical stage and functional class. There was an inverse correlation of the material with serum C4 level. Rheumatoid synovial fluids generally contained higher levels than serum, and five of 23 contained very much higher levels. The frequency of elevated levels of immune complex-like material in sera of patients with systemic lupus erythematosus (2 of 29) and with miscellaneous vasculitides (2 of 21 was much lower than in RA, suggesting that mRF exhibits a specificity for only certain kinds of immune complexes. The reason for this apparent specificity may explain such distinctive features of RA as the high frequency of polyclonal RF, the lack of immune complex nephritis, and the generally normal levels of serum complement.

Mediation of immunologic discharge of lysosomal enzymes from human neutrophils by guanosine 3',5'-monophosphate. Requirement of calcium, and inhibition by adenosine 3',5'-monophosphate.

The purpose of this investigation was to elucidate the relationship of cyclic GMP and calcium to the immunologic discharge of lysosomal enzymes from purified human neutrophils. Contact of neutrophils with a variety of immunologic stimuli, including zymosan particles treated with either normal or rheumatoid arthritic (RA) serum, heat-aggregated (agg) IgG, particulate and immobilized agg IgG each treated with RA serum, and zymosan-treated serum, provoked the discharge of beta-glucuronidase, but not cytoplasmic lactate dehydrogenase, and stimulated the accumulation of cyclic GMP. Both enzyme release and elevation of cyclic GMP levels required the presence of extracellular calcium as neither cellular event proceeded in its absence. Cholinergic enhancement of the immunologic secretion of beta-glucuronidase from neutrophils by acetylcholine was associated with a concomitant accumulation of cyclic GMP. These actions of acetylcholine on neutrophils did not proceed in the absence of extracellular calcium. Whereas the concentrations of cyclic GMP in neutrophils were elevated by both immune reactants and a combination of the latter and acetylcholine, cyclic AMP levels remained unaltered. Thus, cyclic GMP, but not cyclic AMP, was associated with the immunologic and pharmacologic discharge of lysosomal enzymes from neutrophils. Contrariwise, cyclic AMP, but not cyclic GMP, was associated with inhibition of lysosomal enzyme release. For example, dibutyryl cyclic AMP and epinephrine inhibited the release of beta-glucuronidase from neutrophils that was elicited by each of the immune reactants tested. Moreover, cyclic AMP levels in the cells were elevated markedly in every instance that enzyme discharge was inhibited by epinephrine. Epinephrine did not alter the neutrophil concentrations of cyclic GMP at times when those of cyclic AMP were elevated. The data in this report constitute partial evidence that the immunologic discharge of lysosomal enzymes from human neutrophils is mediated or signaled by intracellular cyclic GMP and that calcium is linked to this stimulation of enzyme secretion.

Receptors for aggregated IgG on mouse lymphocytes: their presence on thymocytes, thymus-derived, and bone marrow-derived lymphocytes.

An autoradiographic binding assay employing (125)I-labeled heat-aggregated mouse IgG2b myeloma protein (MOPC 141) was used to demonstrate receptors for IgG on 20-45% of Balb/c thymocytes and on 70-80% of splenocytes. Binding could also be shown with heat or BDB aggregates of another IgG2b (MOPC 195), with IgG1 and with human gamma-globulin, but not with aggregated chicken gamma-globulin, IgA, BSA, nor with aggregated Fab fragments of IgG2b. Optimum binding was obtained at 37 degrees C. Detection of binding was dependent upon aggregate size with complexes of more than 100 IgG molecules being optimal, aggregates of 6-25 detecting splenocytes but not thymocytes, and aggregates of less than 6 binding to a negligible extent. Comparison of grain counts on various cell types showed mastocytoma cells (P815) and macrophages averaging 40-50 grains/cell/day, allogeneically activated thymocytes 20-30, splenocytes 2-3, L5178 lymphoma cells 1, and positive thymocytes 0.6 grains/cell/day. Double labeling experiments for surface Ig, theta-antigen, and agg IgG receptor on mouse spleen cells indicated that a relatively high density of receptor was present on about 80% of B cells, 30% of T cells, and 60% of SIg(-), theta(-), null cells.