Facb Fragment Research Articles

Regulation of human natural cytotoxicity by IgG—I. Characterization of the structural site on monomeric IgG responsible for inhibiting natural killer cell activity

Inhibition of human natural killer (NK) cell activity upon exposure of peripheral blood lymphocytes (PBL) to IgG in monomeric form (mIgG) was found to be dose-, time- and temp-dependent. PBL incubated for 2 hr at 37°C in the presence of myeloma protein of a certain class or subclass had a significant reduction of their NK activity when exposed to IgG, but not to IgM or IgD, and the IgG-induced inhibition of NK cells was observed only when IgG1 or IgG3 paraproteins were used. IgG3 isolated from normal serum had a higher inhibitory property than that of total mIgG. The cytophilic activity of the IgG molecules was confined entirely to the Fc region and seemed to be localized in the C H3 domain, since human and rabbit Facb fragments had a reduced ability to inhibit NK activity. When synthetic peptides representing various sequences of the human γ-chain were tested for inhibition of NK activity, only treatment of effector cells with a peptide comprising the sequence Tyr 407-Arg 416 of the C H 3 domain showed a reduction of NK cell function comparable to the inhibition obtained following incubation of cells in the presence of mIgG. However, on a molar basis, this peptide was 20 times less active than mIgG. In contrast, peptides derived from sequences in the C H 2 domain lacked this inhibitory capacity. Our data indicate that the structural site responsible for inhibiting NK cell activity is located in the C-terminal domain of the IgG molecule.

Lymphocytes bearing Fc gamma receptors in rheumatoid arthritis. III. Immunoregulatory function associated with Facb rosette-forming cells.

A subpopulation of mononuclear cells (PBMNC) that expresses Fc receptors with specificity for the C gamma 2 region of IgG may be detected by rosette formation with calf erythrocytes coated with the Facb fragment of rabbit IgG. These Facb-R+ cells are found in increased numbers in the peripheral blood of patients with rheumatoid arthritis (RA). Studies have been carried out to identify the functional properties of these cells in healthy and rheumatoid subjects. Facb-R+ cells were shown to lack both natural killer and antibody-dependent cytotoxic activity. Depletion of Facb-R+ cells from both healthy and rheumatoid PBMNC resulted in a marked suppression of pokeweed mitogen (PWM) stimulated IgG synthesis but had no effect on T cell proliferation induced by phytohaemagglutinin, concanavalin A, or PWM. The addition of Facb fragments to PBMNC cultures also caused inhibition of PWM-driven IgG production. In this assay rheumatoid PBMNC were significantly less sensitive to Facb-mediated suppression than healthy control cells. Our results suggest that Facb-R+ cells are involved in the antibody-mediated feedback regulation of immunoglobulin synthesis and that this mechanism is impaired in patients with RA.

Quantitative immunoassay for IgA class circulating immune complexes using solid phase Facb fragment of anti-C3

A quantitative assay of IgA class circulating immune complexes (IgA-CIC) by a solid phase anti-C3 enzyme immunoassay (anti-C3 EIA) is described. A stable and reproducible standard for determination of IgA-CIC was prepared successfully by chemical binding of complement C3 to human serum IgA. Two of 27 sera from patients with systemic lupus erythematosus (SLE), however, contained high concentrations of IgA class anti-F(ab′) 2 antibodies that caused false positive results when the F(ab′) 2 of anti-C3 was used for EIA. Solid phase Facb of anti-C3 was found to eliminate the false positive results caused by IgA class anti-F(ab′) 2 and IgA class rheumatoid factor. Good reproducibility and recovery were observed with this Facb anti-C3 EIA using the IgA-C3, a stable standard material, and so this method should be useful clinically in elucidating the role of IgA-CIC.

The interaction of the Facb fragment of rabbit anti-sheep red cell IgG with guinea pig macrophages, and human monocytes and granulocytes

Yasmeen et al ( J. Immun. 110, 1706–1709, 1973) have previously reported on the binding requirements of the guinea pig peritoneal macrophage Fc receptor. The C γ3 domain fragments of human IgGl, in contrast to the C γ2 domain fragment, were able to bind to these macrophages, as demonstrated by both direct and indirect rosette tests. We now report that we have been unable to show binding by the C γ2-bearing rabbit Facb fragment to either peritoneal or alveolar macrophages of the guinea pig. This evidence is therefore in agreement with the hypothesis proposed by Yasmeen et al. (1973) that the C γ2 homology region does not contribute directly to the binding requirements for this cell type. The same protein, rabbit anti-sheep erythrocyte Facb, when coated on sheep erythrocytes. did not form rosettes with human granulocytes. but did form some rosettes with human monocytes.

ISOLATION AND CHARACTERIZATION OF Fcγ RECEPTORS FROM HUMAN PLACENTA

Placental extract was prepared with Tris-HCl buffer pH 7.4 containing EDTA and 2-mercaptoethanol. It agglutinated erythrocytes (E) sensitized with subagglutinating amounts of IgG antibodies from rabbits, guinea pigs and mice, but not E sensitized with IgG from chickens. E or E sensitized with F(ab)2 or IgM antibodies were not agglutinated. The agglutination of EA by the extract was inhibited by human, rabbit and guinea pig IgG, but not by bovine and porcine IgG. Aggregated IgG was more inhibitory than monomeric IgG. IgG3 was the most effective subclass. The extract inhibited the formation of EA rosettes with human mononuclear cells, but did not influence the formation of E or EAC rosettes. The significance of the disulfide bonds and the C gamma 3 and C gamma 2 regions was implied by the finding that the extract neither agglutinated E sensitized with preparations of mildly reduced and alkylated IgG, nor with Facb fragments. These preparations did not inhibit the agglutination. The results strongly indicated that the active component was solubilized placental Fc receptor (FcR). Functionally active FcR was purified by affinity chromatography using aggregated human IgG coupled to Sepharose 4B. SDS-PAGE if the purified FcR under reducing conditions showed one distance band corresponding to approx. 47,000 daltons. The band neither consisted o Ig fragments nor Clq. A rabbit antiserum against the FcR inhibited the agglutination of EA by the extract and stained th FcR-positive areas in placenta.

Tryptic and plasmic cleavage of a rat myeloma IgD

Rat cell surface immunoglobulins were analysed in sodium dodecyl sulfate polyacrylamide gels. By specific immunoprecipitation a 200,000 dalton IgM and a 165,000 dalton IgD were demonstrated. A molecule similar to the mouse and rabbit Fc receptors was non-specifically precipitated. The digestion of a rat myeloma IgD with trypsin or plasmin was studied. Although the IgD molecule was rapidly converted in Fab and Fc fragments by trypsin (1 min., 0°C) it was relatively resistant to plasmin. (Several hours at 37°C were necessary to obtain a complete conversion.) During the plasmin digestion a Facb fragment was observed. These results are discussed in regard to a possible physiological cleavage of cell surface IgD.

The isolation of a globular fragment of rabbit immunoglobulin G probably corresponding to the intact Cγ2 homology region

A compact globular fragment was derived from papain digest of the Facb fragment of nonimmune rabbit IgG. The fragment is a glycoprotein with molecular weight 15250 and its tertiary structure has a substantial amount of β-pleated sheet. Based on its origin, antigenic specificity, sialic acid determination, physicochemical characteristics and functional behaviour, the fragment is most probably the intact Cγ2 domain of rabbit IgG.

Fcgamma receptors in human choroid plexus.

Crysotat sections of human choroid plexus adsorbed erythrocytes sensitized with IgG antibodies of human and rabbit origin. No adsorption occurred when the erythrocytes were sensitized with F(ab')2 or Facb fragments. The reaction was strongly inhibited by intact IgG and by Fc fragments and not inhibited by Facb and F(ab')2 or albumin. These properties are similar to those of corresponding receptors in human placenta. The presence of an Fcgamma receptor in choroid plexus may be of significance for the transfer of IgG from blood to cerebrospinal fluid.

Allotype Suppression in Rabbits: The Requirement for CH3 Domain of Anti-Allotype Antibody

Facb fragments of rabbit anti-allotype antibody were prepared by plasmin digestion and isolated by gel chromatography. The antibody preparation was used in an attempt to induce allotype suppression in newborn rabbits. The Facb fragments were found to be ineffective in inducing the allotype suppression. Administration of Facb fragments caused a "burst" of immunoglobulin synthesis almost immediately after the administration of the antibody. It was concluded that the CH3 domain, which is responsible for the cytophilic activity of the antibody, is essential in induction of allotype suppression.

Circular dichroism studies on proteolytic cleavage fragments from rabbit IgG

The circular dichroism (CD) spectra of proteolytic cleavage fragments derived from rabbit IgG have been studied. In particular, the CD spectra of C γ2 and C γ3 domain-containing fragments have been obtained and compared with their human counterparts. At wavelength below 250 nm, the CD spectra of the Facb, F(ab′) 2, Fc, pFc′ and tFc′ fragments from rabbit IgG were characterized by a negative absorption maximum at 217 nm, indicating the presence of β-pleated sheet structure. The magnitude of this absorption was significantly greater in the spectrum of the F(ab′) 2 fragment. a negative absorption at 255 nm was observed as a shoulder in the CD spectra of intact rabbit IgG and its Fc fragment, but was absent from those of the other fragments, including both the pFc′ and Facb fragments. The structural feature giving rise to the CD transition at 225 nm was shown to be destroyed by plasmin digestion of acid treated IgG, indicating that this transition reflects an interaction between the C γ2 and C γ3 domains of rabbit IgG. A comparison of the CD spectra of human and rabbit Fc fragments revealed conformational similarities, whereas qualitative and quantitative differences in the spectra of the corresponding pFc′ fragments (C γ3 dimer) was observed. The CD spectrum of the rabbit pFc′ fragment was characterized by a negative maximum at 217 nm, whereas the human pFc′ fragments spectrum was characterized by a negative maximum at 225 nm. At wavelengths greater than 250 nm, the CD spectra of both human and rabbit pFc′ fragments were qualitatively similar. Such studies suggest that the human pFc′ fragment, in marked contrast to the rabbit pFc′ fragment, undergoes significant changes in secondary structure upon cleavage from the intact IgG molecule.

Indications That the Cγ2 Homology Region Is Not a Regular Domain

The Cgamma2 homology region of rabbit IgG does not behave like a domain. Thus, there is no trans-interaction between the two Cgamma2 regions; instead there is an unusual cis-interaction between Cgamma2 and Cgamma3 regions. The observations were made on the plasmin digestion products Facb (IgG minus the Cgamma3 region) and pFc' (Cgamma3 region), which did not dissociate under neutral conditions but dissociated in 3M guanidine solution (that is, cis-interaction between Cgamma2 and Cgamma3). The Facb fragment split into subunits with equal molecular weights under neutral conditions on partial reduction and alkylation (that is, lack of trans-interaction between the two Cgamma2 in the molecule).

Specificity of Fcgamma receptors in human malignant tissue and normal lymphoreticular tissue.

The specificity of the Fcgamma receptors in normal spleen and liver and in malignant tissues was studied using hemadsorption to cryostat sections. Indicator cells (EA) were sheep erythrocytes (E) sensitized with rabbit IgG antibody (A). The binding of EA to sections of normal and malignant tissues was inhibited by pooled IgG of human, rabbit, and guinea pig origin and by human IgG1, and IgG3, and IgG4 myeloma proteins. Heat-aggregated IgG inhibited the binding to sections of liver and some malignant tissues more effectively than monomeric IgG. The Fc fragments of IgG were also inhibitory, but not the F(ab')2, Fab', and Facb fragments. The inhibition obtained increased with decreasing amounts of A used for sensitization of E. The inhibitory activity of IgG was abolished after partial reduction and alkylation. No inhibition was obtained with IgG2, IgM, IgA, or albumin. E sensitized with Facb or F(ab')2 fragments of A did not bind to normal or malignant tissues. The specificity of the Fc receptors in normal spleen and liver and in malignant tissues is apparently very similar.

Biologic Activities of Rabbit Immunoglobulin G in Relation to Domains of the Fc Region

Biologic properties of antibodies are known to be mediated by the Fc portion of the H chains. In the present study such properties as complement fixation, cutaneous anaphylaxis, and macrophage cytophilia were examined in relation to the CH2 and CH3 domains of rabbit IgG. Fragments containing but one of these domains were prepared from plasmin and papain digests. Facb fragments of anti-DNP antibodies, together with the antigen DNP-BSA, were able to fix complement by the classical pathway, a result which implicates the CH2 domain; however, guinea pig Fab fragments directed to regions of the rabbit antibody molecule other than CH2 were able to inhibit complement fixation. Facb fragments were unable to mediate PCA or reverse PCA reactions in guinea pigs, nor were CH3 fragments active in tests of reverse PCA or inhibition of PCA. These results suggest that the entire Fc region is needed for cutaneous anaphylaxis. The ability to bind to guinea pig lung macrophages was studied with a rosette technique. Facb fragments were active whereas CH3 fragments failed to inhibit. It is suggested that although some effector functions of antibodies can be assigned to individual domains, others require the entire Fc region.

The effect of acid treatment upon the susceptibility of rabbit IgG to proteolytic cleavage with various enzymes

The pH of solutions of rabbit IgG was adjusted to 2·7 with HCl and incubated at 37°C for 1 hr after which aliquots were adjusted to the required enzyme pH optimum and digested for 4 hr at 37°C with various proteolytic enzymes. The digestion products obtained were compared with those produced under similar digestion conditions but by omission of the acid treatment step. In all cases it was observed that acid-treated IgG was completely digested irrespective of the enzyme used, whereas non-acid treated IgG exhibited varying susceptibility to cleavage by different enzymes. Trypsin, pepsin and papain digestion of acid-treated IgG each produced a major fragment similar in size and antigenic composition to that of F(ab′) 2. None of these fragments retained the capacity to react with rheumatoid factor. Fragments similar to the Facb fragment produced by plasmin digestion of acid-treated rabbit IgG were not observed. The significance of these findings is discussed.

Structural requirements in the Fc region of rabbit IgG antibodies necessary to induce cytotoxicity by human lymphocytes.

Rabbit IgG anti-chicken erythrocyte antibodies were compared with the Fab/c or Facb fragments of IgG and with partially reduced and alkylated IgG for the capacity to induce cytotoxicity by normal human lymphocytes. The Fab/c antibody fragment, which lacks one Fab region, was still able to induce cytotoxicity. In contrast, the Facb antibody fragment, which lacks the C-gamma3 domains, was nearly ineffective in activating the effector cells, whereas intact antibody activity was demonstrated by its ability to inhibit the cytotoxicity induced by unsplit IgG. Similarly, partial reduction and alkylation of the IgG antibodies, under conditions affecting the interchain disulphide bonds only, greatly diminished their ability to induce cytotoxicity, although they effectively inhibited the cytotoxicity induced by untreated IgG. On the basis of these results and previous data, we suggest that the reaction of the Fc region of IgG with the effector cell depends on the integrity of the C-gamma2 domain in the native, divalent state or on the interaction between the C-gamma2 and C-gamma3 domains.

Localisation of monocyte binding site of human immunoglobulin G.

THE binding of IgG to macrophages is thought to be important in both the afferent and efferent limbs of the immune response1–3. Berken and Benacerraf4 studied the properties of guinea pig IgG2 antibodies cytophilic for macrophages and concluded that the binding site was in the Fc region. Similarly Lo Buglio et al.5 and Abramson et al.6 inhibited with isolated Fc fragment the binding of human monocytes to red cells coated with anti-D. Abramson et al.6 also studied the inhibitory activity of various fragments obtained by peptic digestion of IgG. The F(ab′)2 fragment was slightly inhibitory, unlike Fab fragment, and the authors suggested that the binding site was in the N-terminal portion of the Fc fragment (now known as the CH2 region). Huber and Fudenberg7 reached similar conclusions following experiments in which F(ab′)2 fragments of anti-D antibodies were attached to red blood cells and produced rosettes with human monocytes, although the possibility of contaminating IgG could not be eliminated. In contrast, MacLennan et al.8 were unable to inhibit neutrophil phagocytosis of sensitised bacteria with immune complexes of antigen and the Facb fragment of rabbit IgG. Since the Facb fragment includes the Fab and CH2 regions but lacks the CH3 region this suggests that the latter may be the site of neutrophil binding. Furthermore, Yasmeen et al.9, working with a heterologous system (human immunoglobulin G and guinea pig macrophages) have presented evidence that macrophage binding is a property of the CH3 region of the molecule. In view of these conflicting reports we have investigated the possible role of the CH3 region of human IgG in binding to homologous monocytes.

Biological activities associated with the Facb fragment of rabbit IgG

Various types of rabbit IgG Fc sub-fragment have been examined for a capacity to fix to heterologous (guinea-pig) skin and to react with rheumatoid anti-IgG-globulins. Facb fragments proved, somewhat surprisingly, to be inactive in both systems; thus resembling pFc′ and tFc′ fragments (derived from the C-terminal end of the Fc region) and in contrast to the active parent Fc fragment. The effect of acid treating whole IgG in a similar way to that employed in preparing Facb fragments was investigated. This treatment per se was found to result in no appreciable loss in biological activity. The demonstration that fragments incorporating the C H 2 or C H 3 structural domains are both inactive suggests the necessity for strict conformational integrity in the expression of at least some biological activities associated with the Fc regions of IgG molecules.

Biological activities associated with the Facb fragment of rabbit IgG

Biological activities associated with the Facb fragment of rabbit IgG