Fc Fragment Of Human IgG1 Research Articles

FC fragments of human IGG may influence allergic reactions

The antiallergic effect of Fc fragments prepared from human polyclonal IgG was investigated in several experimental models. In an in vitro assay, isolated ileum of cynomolgus monkeys was sensitized with serum from atopic patients. In six of fifteen monkeys the subsequent addition of specific allergens reproducibly resulted in an ileum contraction measured in the Schultz - Dale apparatus. In all six positive monkeys pre-treatment of the isolated ileum with Fc (papain) before sensitization inhibited ileum contraction. Fc (plasmin) or Fc (pepsin) was less or not effective, aggregated IgG, monomeric IgG, sulfonated IgG, the Fc-free F(ab') 2 moiety, or albumin had no significant or no reproducible inhibitory effect. In an in vivo assay passive cutaneous anaphylaxis was studied in 28 cynomolgus monkeys. Different dilutions in PBS of the particular specific allergens were subsequently administered to sensitized skin sites, simultaneously to i.v. injection of Evans blue. The degree of the local allergic (passive cutaneous anaphylactia) reaction was evaluated by measuring the diameter of the blue area at the dermal injection sites. About 50% of sera induced positive reaction in monkeys. Compared to the control (application of PBS), the degree of the positive reactions could be reduced by about 50% if Fc (papain) (optimal dose 25 mg/kg body weight) was injected i.v. either 2 h before or after local sensitization. No significant inhibitory effect could be found with the Fab moiety of IgG. Follow-up studies showed that the inhibitory effect of Fc lasted for about 10 days. The pharmacological basis of the observed antiallergic action of Fc is not yet understood.

Antagonistic regulatory properties of the Fc region of immunoglobulin

Coculture of human peripheral blood mononuclear cells with Fc fragments of human IgG, or the synthetic Fc region-derived peptide, p23, results in the release of oxidative products of arachidonic acid. Prostaglandin E was the major arachidonic acid metabolite found in the culture supernatants. Induction of polyclonal antibody production by Fc fragments and p23 is influenced by the concomitant production of prostaglandin E in culture. Addition of prostaglandin synthetase inhibitors, indomethacin and aspirin, to human peripheral blood mononuclear cell cultures resulted in a significant increase in the amount of polyclonal antibody produced. Moreover, addition of exogenous prostaglandin E to these cultures abrogated the ability of indomethacin to enhance Fc fragment-induced polyclonal antibody production. These results suggest that Fc fragments possess bifunctional immunoregulatory properties.

Monoclonal opsonic mouse antibodies specific for streptococcal IgG Fc-receptor.

Spleen cells from mice immunised with group-A type-M15 streptococci and boosted with purified IgG Fc-receptor (FcR) from this type were fused with Sp 2/0 mouse myeloma cells. The resulting hybridomas were screened by ELISA for antibody production. Two IgM-secreting cell lines were selected. The monoclonal antibodies and ascites fluids inhibited the binding of 125I-labelled human IgG and IgG Fc-fragments to group-A type-M15 streptococci. The monoclonal antibodies also displaced purified FcR towards the anode in electrophoresis. They opsonised group-A type M-15 streptococci for phagocytosis by human granulocytes in the presence of fresh human serum. It was concluded that FcR is important for group-A streptococcal virulence.

Immunological demonstration of intestinal absorption and digestion of protein macromolecules in the trout (Salmo gairdneri).

An immunofluorescence technique using antibodies against the Fc and Fab fragments of human IgG (IgGH) was used to study the absorption of proteins by the intestinal epithelial cells of rainbow trout after oral or anal administration. Cellular absorption of a high molecular weight protein, hepatitis-B surface antigen (HBsAg), was also studied by using two monoclonal antibodies, one specific for the confirmation of the antigen (implying disulfide bridges), and the other that reacts with the constituent polypeptides. Both absorbed IgGH and HBsAg were seen to be segregated in the apical vacuolar system, a characteristic feature of intestinal epithelial cells. The same antibodies were used with an everted sac technique in conjunction with immunofluorescence, to show the intravacuolar degradation of IgGH and HBsAg following absorption. By using an antibody against cathepsin D, it was possible to demonstrate, by immunofluorescence, the localization of this enzyme in the same vacuolar system. After coupling the antibody to peroxidase or to the protein A/colloidalgold complex, the ultrastructural antigenic sites of cathepsin D could be seen to be localized in the interior of the vacuoles. The vacuolar localization of a cathepsin B activity was determined by incubating sections of intestinal mucosa, or isolated epithelial cells, with a specific synthetic substrate (Z-Ala-Arg-Arg-methoxynaphthylamide). The supranuclear hyaloplasmic vacuoles of intestinal epithelial cells may be considered to be phagolysosomes that assure the degradation of absorbed proteins. This function may be of fundamental importance in the in the nutritional processes of this species.

The immunomodulatory effect of human IgG Fc fragments on the oxazolone-specific immune response of high and low responder mice

Intravenous injection of human IgG Fc fragments in mice resulted in the stimulation or inhibition of an oxazolone-specific antibody response depending on the schedule of Fc fragment injection. High and low responder mice for oxazolone were injected with Fc fragments according to two protocols: either on the day of oxazolone priming, or together with the oxazolone boost, and the isotype composition of oxazolone-specific antibodies was analysed by solid phase radioimmunoassay. We found the primary and secondary anti-oxazolone IgM levels increased in all instances, irrespective of the schedule of Fc fragment treatment. In contrast, the oxazolone-specific IgG production was increased only if Fc fragments were injected at the time of antigen priming. Injection of Fc fragments together with a secondary injection of oxazolone resulted in the inhibition of oxazolone-specific IgG production. Both stimulation and inhibition of oxazolone-specific antibodies were more pronounced in the low responder C57BL/6 mice strain.

Human-human hybridoma autoantibodies with both anti-DNA and rheumatoid factor activities.

Human hybridomas have been produced by fusing peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with the GM 4672 human cell line. 262 hybridoma clones from the fusions of four RA and five SLE patients were screened for binding to denatured DNA (dDNA), native DNA, and the Fc fragment of human IgG (HIgG). Of the 17 hybridoma antibodies (nine RA, eight SLE) selected for strong binding to denatured DNA, Fc, or both, five reacted with dDNA only, one with Fc only, and eight with both dDNA and Fc. Hybridoma supernatants exhibiting dual reactivity were absorbed over HIgG and bovine serum albumin (BSA)-Sepharose immunoabsorbent columns. The reactivities to both DNA and HIgG were completely removed by the HIgG column but unaffected by passage over the BSA column, and both DNA binding and rheumatoid factor activities were recovered in the acid eluates from the Sepharose-IgG column. The binding of dual reactive hybridoma autoantibodies to the Fc fragment of HIgG was specifically competed by dDNA and HIgG, providing additional evidence that one antibody may be capable of reacting both as a rheumatoid factor and as an anti-DNA antibody.

Cyanogen bromide cleaves Fc fragments of pooled human IgG at both methionine and tryptophan residues

An attempt was made to obtain fragments containing the Cγ 2 region by selectively cleaving human Fc fragments prepared from pooled IgG at Met residues using cyanogen bromide. Based on the known locations of Met residues in the Fc regions of human IgG subclasses, fragments between Met 252 and 358, comprising the Cγ2 domains, were expected from IgGl Gm — 1, IgG2, IgG3 and IgG4. Greater fragmentation of the Fc fragments occurred, however, than was predicted. Automated N-terminal sequencing identified five major points (Trp 381, 313 and 277, and Met 397 and 252) and two minor points of cleavage (Met 428 and Trp 417). The majority of cleavage points occurred at Trp rather than Met. Furthermore, cleavage at Met 358, necessary to produce Cγ 2 domains, was not detected. Control experiments verified the integrity of the Fc fragments handled in exactly the same manner without cyanogen bromide exposure and the ability of the same cyanogen bromide preparation to produce the expected cleavages at Met of sperm whale apomyoglobin without fragmentation at Trp. Cleavage at Met 358 did not occur presumably because of the difficulty associated with cyanogen bromide cleavage at Met-Thr peptide bonds. Cleavage at Trp probably occurred by way of halogen promoted oxidation of the indole nucleus with resultant peptide bond fissure. These observations show that cyanogen bromide cleavage of pooled human Fc fragments is not selective for Met, but also cleaves at Trp residues. The resultant fragmentation of the Cγ 2 region coupled with the inability to make the required cleavage at the 358–359 Met-Thr bond resulted in the inability to produce fragments comprising the Cγ2 domains. The reasons for the selective cleavage at Met in some proteins and the cleavage at both Trp and Met in others are not known.

Bifunctional lymphocyte regulation by human Fcγ fragments and a synthetic peptide, p23, derived from the Fc region

Fc fragments of human IgG 1 and the synthetic peptide, p23, representing residues 335–357 in the CH 3 domain of IgG 1 were able to increase levels of secreted Ig in murine spleen cell cultures. B cell activation by Fcγ fragments was macrophage- and T cell-dependent whereas activation by p23 was only T cell-dependent. Induction of Ig secretion by both stimulators was influenced by endogenous oxidative products of arachidonate, as evidenced by the augmentation of Ig levels in cell cultures treated with indomethacin (IM), a prostaglandin (PG) synthetase inhibitor. Both Fcγ fragments and p23 were able to induce the release of PGE from splenic adherent macrophages and, in the former case, the release was inhibited by either IM or aspirin. Moreover, addition of either exogenous PGE 1 or PGE 2 reduced the levels of secreted Ig in Fcγ fragment- or p23-stimulated cell cultures. These data suggest that B cell activation by Fcγ fragments is influenced by the concomitant induction of suppressive PG.

Anti-immunoglobulin antibodies IV. Cross-reaction of anti-idiotypic antibodies specific for rabbit and murine anti-a1 allotype antibodies with Fc fragment of human immunoglobulins.

Anti-idiotype (Id) antibodies against anti-a1 rabbit allotype antibodies were produced in rabbits and mice by immunization with polyclonal rabbit anti-a1 allotype or with a monoclonal murine anti-a1 allotype antibody, respectively. These antibodies recognize an interspecies cross-reactive idiotope on anti-a1 allotype antibodies. However, these anti-Id antibodies also react with various subclasses of human IgG. In multiple systems, it was shown that these anti-Id antibodies interact with the Fc fragment of human IgG and, therefore, exhibit a rheumatoid factor-like activity. These results support the hypothesis that gamma-globulin may not be the only stimulus to clones producing rheumatoid factors and that perhaps other antibodies, especially anti-Id antibodies, also bind gamma-globulin secondarily.

The internal image of IgG in cross-reactive anti-idiotypic antibodies against human rheumatoid factors.

A network of idiotypes and anti-idiotypes has been hypothesized to modulate antibody production against exogenous antigens. Idiotypic antigens on autoantibodies have been studied because of their potential use for specific immunomodulation. The present studies describe the preparation and characterization of rabbit anti-idiotypic antibody against human IgM anti-IgG autoantibodies (rheumatoid factors, RF) that bear the "internal image" of the human IgG-Fc fragment, and hence react specifically with the majority of RF from patients with rheumatoid arthritis. The anti-idiotype was isolated from rabbit anti-RF antisera by either immunodepletion of anti-immunoglobulin antibodies, or more simply by a single affinity purification step on a rabbit anti-human IgG Fc column. As measured by an enzyme-linked immunoassay, the anti-idiotype prepared by both methods bound to plates coated with purified IgM RF, but not to plates coated with non-RF IgM proteins. The anti-idiotype dose dependently blocked the binding to IgG of IgM-RF in 83% of sera from multiple patients with rheumatoid arthritis, Sjogren's syndrome, and macroglobulinemia. The anti-idiotype did not inhibit the activity of human IgM antibodies against DNP, tetanus toxoid, or thyroglobulin. The antigen recognized by the cross-reactive anti-idiotype was not apparently associated with a particular light or heavy chain amino acid sequence, but rather was intrinsic to most immunoglobulins with RF activity. Broadly cross-reactive anti-idiotypes with the "internal image" of IgG are simple to generate, and react with most RF. They may facilitate studies on the specific regulation of the human anti-IgG autoantibody response.

Direct tissue isoelectric focusing of nervous system and muscle sections for detection of IgG patterns.

Agarose isoelectric focusing (IF) of 5-70 microns sections of frozen autopsy material (direct tissue IF; DTIF) from the central nervous system (CNS) and peripheral nervous system (PNS), and from muscle biopsy, followed by immunofixation with unlabeled or radiolabeled anti-serum against human gamma chains or IgG Fc fragments has been developed for analysis of IgG patterns. The DTIF technique followed by antiserum immunofixation is a promising tool for characterization of proteins in autopsy and biopsy materials from nervous and muscular systems. In multiple sclerosis brains, oligoclonal IgG bands were found which differed regarding number and mobility between the brains, and also between two different regions in single brains. For PNS, IgG patterns were easily detectable. In polymyositis muscle, no difference was found for IgG patterns when compared with normal muscle or serum.

Properties of Fc gamma receptors in the human central nervous system.

Cryostat sections of human central nervous system (CNS) tissue absorbed sheep erythrocytes (E) sensitized with rabbit IgG antibody (A). The indicator cells bound to the choroid plexus, to the leptomeninges covering the brain and spinal cord, to the arachnoid granulations and to the perivascular tissue of the neural parenchyma. Unsensitized E or E sensitized with F(ab')2 fragments of IgG were not bound. The reactions were inhibited by pooled human IgG, human IgG1 and IgG3 myeloma proteins, and Fc fragments of pooled human IgG. Whole human IgG2 myeloma protein, IgM, IgA, F(ab')2 fragments of pooled human IgG and albumin did not inhibit. Experiments with reduced and alkylated IgG, EDTA, iodoacetamide, 2-mercaptoethanol, various pHs and salt concentrations, formaldehyde, periodic acid and neuraminidase did not reveal any differences between the Fc gamma receptors in the separate anatomical areas. The Fc gamma receptors in human CNS are thus apparently very similar.

A simple plaque assay method for antibody-dependent cell-mediated cytotoxicity using Cunningham's chamber

An improved and simpler plaque assay method for antibody-dependent cell-mediated cytotoxicity using Cunningham's chamber has been developed. Effector lymphocytes, target sheep red blood cells (SRBC) and anti-SRBC serum were incorporated together into a Cunningham's chamber and, immediately after making monolayers, incubated at 37°C for 20 h. A hemolytic plaque of SRBC was formed around a lymphocyte in the presence of anti-SRBC serum, which was inhibited specifically by addition of aggregated IgG or Fc-fragment of human IgG in the culture. A hemolytic plaque formed around a monocyte-like cell was clearly differentiated from those around a lymphocyte (K-cell). The percentage of plaques in the purified lymphocytes from healthy individuals correlated significantly with ADCC activity measured by 51Cr-release. The method is a significant improvement on the earlier plaque assay methods with regard to: (1) making the monolayer, (2) observation and counting of plaques, and (3) shortening the time required for assay. Thus, the method should be valuable for clinical studies of cytotoxic activity in ADCC systems in healthy and diseased persons.

Activation of T lymphocytes by the Fc portion of immunoglobulin.

T lymphocytes are stimulated to release T-cell-replacing factors in response to Fc fragments of human IgG, Lyt 1+23- T cells are directly triggered to factor production by Fc subfragments, derived from intact Fc fragments by macrophage-dependent enzymatic cleavage. These factor(s) replace T cell function in two Fc-mediated immune responses; induction of polyclonal antibody synthesis, and potentiation of anti-SRBC responses.

Monitoring the carbohydrate component of the Fc fragment of human IgG by 13C nuclear magnetic resonance spectroscopy

Monitoring the carbohydrate component of the Fc fragment of human IgG by 13C nuclear magnetic resonance spectroscopy

A direct binding assay for rheumatoid factor serum antiglobulins using fluorescein-labelled Fc fragment of human immunoglobulin-G.

A simple routine method is described for the quantitation of antiglobulin (rheumatoid factor) in human serum irrespective of its immunoglobulin class. Papain Fc fragment of human IgG is labelled with fluorescein isothiocyanate and incubated with the serum. Bound Fc is separated from free Fc by precipitation with polyethylene glycol and measured by the fluorescence of the precipitate. Results are expressed as a percentage of the binding obtained with a normal pool serum. About 60% of seronegative rheumatoid sera and 96% of seropositive rheumatoid sera gave results more than 2SD above the mean value for normal sera. Only 17% of osteoarthritis sera gave positive results by the same criterion. A relationship is also deduced to show that if the test is carried out at a number of Fc concentrations a double reciprocal plot of bound Fc against free Fc enables both the total antiglobulin and the ratio of binding constants of antiglobulin for Fc and for IgG to be calculated. This ratio is approximately unity. Under the standard conditions the results correspond to a measurement of approximately 30% of the total antiglobulins present.

Separation of IgG Fab and Fc fragments by isoelectric focusing

Preparative liquid isoelectric focusing in a sucrose stabilized gradient was employed to separate Fab and Fc fragments of human and chicken IgG. Immunoelectrophoresis was used to confirm separation of the fragments. The advantages of isoelectric focusing over the classical method of ion exchange chromatography are discussed.

Radioimmunoassay of IgG and IgM Rheumatoid Factors Reacting with Human IgG

Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation.

B-lymphocytes activation by the Fc region of IgG.

Strong stimulation of DNA synthesis (up to 150-fold) and blast transformation can be induced in mouse spleen cells by Fc fragments of human IgG. The mitogenic response is optimal on day 5 of culture and is dependent on the concentration of Fc fragments with a sedimentation rate of 3-5S. Intact IgG is also stimulatory, but only when modified by heat aggregation, and produces only a 10-fold increase in [3H]thymidine uptake. The stimulation by aggregated IgG is dependent on the Fc portion, since aggregated (or soluble) Fab or F(ab')2 fragments are inactive. The results show that the response is T-cell independent and that it is a function of nylon wool adherent, surface Ig-positive, Fc receptor-bearing B lymphocytes. Fc fragments do not induce plaque-forming cells to human IgG in normal mouse spleen cell cultures, but rather trigger polyclonal antibody synthesis (anti-goat erythrocytes, anti-2,4,6-trinitrophenyl). It is postulated that the Fc region of antibodies plays a role in the regulation of the humoral immune response by triggering clonal expansion of B lymphocytes.

Purification of immunolobulin G Fc-reactive factor from Streptococcus azgazardah.

A factor from Streptococcus azgazardah (Lancefield's group C) capable of reacting with the Fc fragment of human IgG was partially purified and characterized. The bacteria were lysed by infection with phage, and the IgG Fc-reactive factor was purified from the crude lysate by affinity chromatography on a IgG-coupled Sepharose 4B column. The column was eluted with 3 M KSCN and the eluate gel-filtered on a Sephadex G 75 column. A 30-fold purification was obtained. The molecular weight of the IgG Fc-reactive factor was estimated to be 60,000. The factor was sensitive to trypsin treatment and to heating 95 degrees C, 10 min at pH 2.0. On polyacrylamide electrophoresis only one band was obtained. The trypsin-and heat-sensitivity, and the estimated molecular weight of the factor was similar to an IgG Fc-reactive factor preparation obtained from group A, type M 56 streptococci by heat treatment at 120 degrees C for 30 min.