Specificity of Receptors for IgG on Human Lymphocyte-Like Cells

Some human lymphocyte-like cells (EA-RFC) have receptors for IgG demonstrable by their ability to form rosettes with human Rh-positive O erythrocytes sensitized with anti-CD isoantibodies (Ripley). The specificity of these receptors for the various Ig classes, IgG subclasses, and fragments of the IgG molecule was determined by studying the inhibitory capacity of the corresponding immunoglobulins in the rosette assay. The receptors showed specificity only for IgG among the Ig classes and about equal affinity for IgG1 and IgG3, but only weak binding of IgG2 and IgG4 was obtained. Whereas no inhibition was obtained with Fab and F(ab')₂ fragments prepared from IgG, the Fc fragment showed strong inhibitory capacity, which was even surpassed by the IgG3 Fch fragment, containing an extension from the N-terminal part of Fc. The inhibitory capacity of the Fc and Fch fragments was considerably reduced by partial reduction and alkylation. The pFc' fragment of IgG, which corresponds to the C_γ3 region, did not inhibit rosette formation. These data indicate that mainly the C_γ2 region is involved in the binding of IgG to EA-RFC. Inhibition studies did not show any differences in the relative inhibitory capacity of monomerie, dimeric, or highly polymerized (heat-aggregated) IgG. However, antibodies of rabbit origin complexed with antigen (ferritin) gave stronger inhibition than the corresponding native Ig.     

Comparison of Fch and Fc Fragments of IgG3: Evidence of an Intra-Chain S-S Bridge in the Hinge of γ3 Chains

The Fc and Fch fragments of human IgG₃, appearing late and early during papain digestion, respectively, were antigenically and structurally compared. Only the Fch fragment reacted with an anti-Fh (hinge region-specific) antiserum; thus the main portion of the hinge region is split off during the formation of Fc fragments The Fch fragment has a higher content of inter-chain S-S bridges than the Fc fragments. The Fc fragments are probably a mixture of three related fragments. The Fch fragment appears to be more homogeneous, although there was hydrolysis at the C-terminal site of the hinge region in some of the Fch chains. Evidence is presented that the γ³ chain bridge has an extra intra-heavy-chain disulphidc bridge at the C-terminal end of the hinge region.     

Isolation of a Fragment, Fh, Corresponding to the Hinge Region of Human IgG3

An Fh fragment corresponding to the hinge region of IgG₃ has been isolated by J single α-chymotrypsin treatment of whole IgG₃ Various combinations of two enzymes have also been used, but with somewhat lower yield and less purity The isolated Fh fragment has features similar to those of the bound hinge region in the intact IgG₃ and its F(ab ' )₂ and Fch fragments. The Fh fragment has a char acteristically high content of cystine and proline and seems to lack aromatic amino acids     

Cleavage of the Four Human IgG Subclasses with Cathepsin G

Cathepsin G, the chymotrypsin-like serine proteinase from human polymorphonuclear leucocytes, cleaves human IgG. The relative susceptibilities of the four IgG subclasses to the action of this enzyme were studied kinetically and showed the following decreasing order of susceptibility: IgG3≫ IgG4>IgG1>IgG2. IgG1 and IgG2 produced primarily F(ab')s and traces of Fc-related fragments. IgG4 gave rise to both Fab and F(ab')₂ as major products, and small amounts of an Fc-related fragment were detected. The cleavage of IgG3 produced various fragments, depending on the experimental conditions: The primary fragments were Fab and Fch (Fc covalently joined to the extended hinge-region polypeptide of IgG3) and an intermediate Fab-Fch species. Both Fab and Fch were further degraded by cathepsin G. Fch was gradually split, giving rise to three subfragments that were finally degraded to dialysable peptides. The enzyme further cleaved the Fab fragment in the heavy-chain portion and released a polypeptide probably representing the V_H domain.     

Three New Fragments, F(ab)2, F(c)2, and Fab/c, Obtained by Papain Proteolysis of Normal Human IgG.

Three new fragments, each with a molecular weight of about 100,000, were isolated after papain proteolysis of normal monomer human IgG. The fragments isolated were as follows: F(c)₂ fragment with Fc determinants only, Fab/c fragment with both Fc and Fab determinants, and F(ab') fragment with only Fab determinants. The F(c)₂ fragment appeared to be a dimer of Fc stabilized by disulphide bonds, whilst the F(ab)₂ fragment consisted of Fab subunits mainly held together by non-covalent forces. The Fab/c fragment is probably a single Fab fragment and the Fc fragment held together by an unbroken heavy chain.     

Alternative Non-Immune F(ab')2-Mediated Immunoglobulin Binding to Group C and G Streptococci

We tested 140 bacterial strains representing 19 different species for binding or purified radiolabelled F(ab')₂ fragments prepared by pepsin digestion of polyclonal and monoclonal human IgG. Both polyclonal and monoclonal F(ab')₂ fragments showed positive binding to group C and G streptococci with maximum uptake levels of 50% and 85%. Binding was obtained both with fresh bacteria and with organisms stabilized by heat treatment. F(ab')₂ fragments of two human IgG1 myeloma proteins with anti-staphylolysin specificity showed a similar binding pattern. IgG present in normal human serum inhibited the uptake of F(ab')₂ fragments, whereas albumin and fibrinogen and purified Fc fragments prepared by papain digestion of polyclonal IgG and monoclonal IgG1 did not show such capacity. Fourteen human myeloma proteins representing IgA, IgM and the four IgG subclasses were tested for inhibiting capacity. Reactivity was noted with at least one myeloma protein within each IgG subclass but not with IgA or with IgM monoclonal proteins. Normal rabbit serum was as inhibitory as normal human serum, whereas dog serum was less reactive. These data demonstrate that group C and G streptococci carry a heat-stable surface component interacting with the F(ab')₂ portion of the IgG molecule. The results suggest that the reactive site on the immunoglobulin molecule may reside in the more constant part of the variable domain. This new reactivity is different from the previously known non-immune reaction involving the IgG Fc portion. This alternative non-immune reactivity is analogous to but distinct from the alternative protein A reaction in Staphylococcus aureus .     

Binding of Fragments of Human IgG to Solid-phase C3b Measured by Enzyme-linked Immunosorbent Assay (ELISA)

The binding of human IgG and different fragment of IgG to C3b adsorbed in polystyrene tubes has been studied by the enzyme-linked immunosorbent Heat-denatured polyclonal IgG and F(ab')₂ and Fab fragments of IgG bound to solid-phase C3b Heat-denatured Fc fragments of IgG also had some reactivity with C3b, but at significantly higher concentrations than F(ab')₂ and Fab fragments. The binding of heat-denatured IgG could not be completely inhibited by the addition of heat-denatured F(ab')₂ fragments in tenfold excess The results suggest that the binding of heat-denatured IgG to solid-phase C3b is mediated through the Fab and Fc portions binding of denatured IgG to solid-phase C3b is mediated through the Fab and Fc portions of IgG molecules.     

Inhibition of Immunoglobulin Production In Vitro by IgG and F(ab')2 Fragments, but not by the Fc Portion

Antibody-secreting B cells were measured as plaque-forming cells (PFC) in a modified haemolysis-in-gel assay, using protein A coupled sheep erythrocytes as targets. Human lymphocytes from blood (PBL), bone marrow or spleen were stimulated in vitro by various polyclonal B-cell activators and incubated with intravenous immunoglobulin (IVIG) or peptide fragments of IVIG. IgG and IgM production from PBL and bone marrow cells, measured as PFC, was inhibited more than 50% by IVIG 2.5mg/ml, compared to controls without IVIG. Inhibition of the IgG and IgM response of spleen cells by IVIG varied depending on the stimuli. Using Staphylococcus aureus protein A (SPA), inhibition was almost 90% ( P <0.001). The inhibition of the IgG and IgM responses to lipopolysaccharide from Escherichia coli (LPS) were 70% ( P <0.01) and 28% ( P <0.05), respectively. IgG stimulation by pokeweed mitogen (PWM) was inhibited by 57% ( P <0.01), but the IgM response was inhibited only by the higher IVIG concentration of 5.0mg/ml. In mixed lymphocyte cultures of spleen cells, IgG and IgM production were inhibited by more than 60% ( P <0.05). The effect of IgG, IgG-F(ab')₂ and IgG-Fc on LPS or PWM-stimulated spleen cells were compared, using equimolar concentrations of the various preparations. IgG- and IgM-producing PFC were significantly ( P <0.05) inhibited in a dose-dependent fashion by IgG and F(ab')₂, but not by Fc. LPS-induced IgG and IgM production was inhibited also when IgG and F(ab')₂ were added up to 48h after the stimulator. A comparison of IgG, F(ab')₂ and Fc products from different companies showed that all IgG and F(ab')₂ preparations significantly inhibited IgG and IgM production of LPS-stimulated spleen cells. No significant inhibition was obtained with any of the purified Fc products.     

Kinetics of the Different Susceptibilities of the Four Human Immunoglobulin G Subclasses to Proteolysis by Human Lysosomal Elastase

Human lysosomal clastase cleaves human monoclonal IgG into components that closely resemble the fragments produced by papain digestion IgG1 produced Fab, Fe and Fab-Fc fragments; cleavage of IgG2 produced Fab-Fc, Fab and Fc fragments: IgG3 gave rise to almost pure Fab und Fch (Fc covalently Joined to the extended hinge region polypetide of IgG3). and from IgG4, F(ab)₂, Fab and Fc fragments were recovered. The relative susceptibilities of the four human IgG subclasses to proteolytic attack by elastase were studied kinetically and showed the following decrease order of susceptibility: IgG3 >IgG1 >IgG2 >IgG4. The Fab fragment from papain digestion of IgG1 and the corresponding fragment from clastase digestion showed indistinguishable molecular weights and immunochemical identity     

Further Characterization of the Alternative Protein-A Interaction of Immunoglobulins: Demonstration of an Fc-binding Fragment of Protein A Expressing the Alternative Reactivity

Intact IgG and fragments F(ab')₂γ, Fabγ and Fcγ from a rabbit anti-protein-A serum (RapA) and corresponding preparations from normal rabbit IgG (NRG) were tested for their inhibitory effect on the binding of protein-A-reactive ¹²⁵I-IgE and ¹²⁵I-Fcγ, respectively, to protein-A–Sepharose. Intact IgG, F(ab')₂γ and Fabγ of RapA inhibited the binding of protein-A-reactive ¹²⁵I-IgE, whereas only intact IgG and Fcγ fragments from both RapA and NRG inhibited the binding of ¹²⁵Fcγ to protein A-Sepharose. Further, the functional relationship between RapA and human polyclonal IgG was studied in a nephelometric test system. Intat IgG or fragments of IgG from human polyclonal IgG and rabbit anti-protein-A were found to affect the precipitation between human IgG and protein A in a similar way. Thus F(ab')₂γ fragments and intact IgG enhanced the precipitation, whereas Fabγ and Fcγ fragments inhibited the precipitation. Protein A and an Fc-binding fragment of protein A (fragment B) were tested for their abilities to link different radiolabelled immunoglobulin preparations expressing the alternative and the classical protein-A reactivity to immobilized Fc fragments. All proteins expressing the alternative reactivity were efficiently bound both by fragment B and by protein A, indicating that fragment B, in addition to its classical Fc-binding activity, also expresses the alternative protein-A reactivity.     

Specificity of Fcγ Receptors in Human Malignant Tissue and Normal Lymphoreticular Tissue

The specificity of the Fcγ 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.     

Interactions of porcine IgG and porcine lymphocytes with protein-A Sepharose

Interaction of Protein-A Sepharose with porcine IgG showed that the binding capacity for porcine IgG was slightly lower than for human IgG. A small proportion of antibody activity of porcine serum was not reactive with Protein-A Sepharose. The interaction occurred with Fc fragments obtained either by papaĭn or by trypsin splitting of IgG but not with Fc' fragments. However, Fab fragments obtained after papaĭn or trypsin hydrolysis as well as Fab'₂ and Fab' fragments obtained by pepsin cleavage were partly retained onto the Protein-A Sepharose column. No antigenic differences were observed between retained and non-retained Fab, Fab' and Fab'₂ fragments with specific anti-porcine light chain antiserum. Thus, the interaction between Protein-A from S. aureus and porcine IgG is mediated not only through the Fc fragment, but also partly through the Fab part of the molecule. The resolubilized heavy chains did not bind to Protein-A Sepharose, suggesting that structural requirements are necessary for recognition. A subpopulation of porcine lymphocytes could be adsorbed onto Protein-A Sepharose beads, a binding specifically inhibited in the presence of porcine IgG.     

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 Cγ3 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 Cγ² domain in the native, divalent state or on the interaction between the Cγ² and Cγ³ domains.     

Prolonged In Vivo IL-4 Treatment Inhibits Antigen-Specific IgG1 and IgE Formation

IL-4 is obligatory for primary IgE responses, whereas primary IgG, and secondary IgE responses are partially IL-4 independent. To investigate the effect of IL-4 on the antigen-specific memory formation for these isotypes, BALB/c mice were treated after primary TNP-KLH immunization with recombinant IL-4 for a period of 4 months. This prolonged presence of a high IL-4 level resulted in increased serum levels of total IgG₁ and IgE, whereas total IgG_2a did not change. The expression of CD23, but not J-A^d, increased on the splenic B cells. IL-4 treatment did not affect the IL-4 production by Con A stimulated spleen cells, whereas it did decrease the IFN-γ production. In the same mice the TNP-specific IgG₁ and IgE serum levels, however, were decreased. Similar results were found when the antigen was continuously present during the IL-4 treatment. Furthermore, it was shown that IL-4 decreased the formation of IgG₁, and IgE memory cells. These results point to different effects of IL-4 in regulating antigen-specific and bystander responses.     

Inhibition of Neutrophil Chemotaxis by Elastase-Generated IgG Fragments

IgG1 is cleaved in vitro by granulocyte elastase into Fc and Fab fragments. The elastase-specific Fc fragment has been previously detected in vivo. Biological activity of the fragments has been described in modulating neutrophil oxidative metabolism and enzyme release. To investigate further effects granulocyte chemotaxis (CT) was tested. The CT was assayed in Boyden chambers and the chemotactic index (CI) was calculated which represents the mean distance travelled by the activated cells. Stimulation of leucocyte CT by casein, activated serum and FMLP gives maximal values of delta CI = 46.7, 26.4 and 7.2 microns, respectively. Native IgG1 and the elastase-produced IgG fragments do not stimulate leucocyte CT. FMLP-stimulated CT is specifically inhibited by the elastase-produced Fc fragments. Addition of 7 nmol Fc to stimulus concentrations of 16 to 125 nM FMLP results in total inhibition of chemotaxis demonstrated by CI values which are lower than those for unstimulated cells. The inhibition of CT is concentration dependent in the range of 2 to 7 nmol Fc/10(6) PMN. Number and affinity of FMLP receptors are not influenced by Fc fragments, so Fc binds neither to FMLP nor the FMLP receptor. CT stimulated by casein shows a large portion of chemokinesis. Only at suboptimal casein concentrations do Fc and IgG have an inhibitory effect on CT (0.63 mg casein/ml, 10 nmol peptide/10(6) PMN). C5a-stimulated CT is not influenced by IgG or IgG fragments which indicates that the samples are not cytotoxic. So the FMLP and casein-stimulated CT is specifically inhibited by the elastase-produced Fc fragments in a low concentration range.     

Immunoglobulin Receptors on Mouse Mast Cells

Interaction of mouse immunoglobulins with a mast cell tumor HC was studied by the rosette technique. All mast cells formed rosettes, and this could be inhibited by IgG₁ myeloma proteins, less efficiently by IgG₂, and not by IgA or IgM. Antigen antibody complexes formed under appropriate con ditions also inhibit, with at least 100-fold greater efficiency than free im munoglobulin. Antisera containing reaginic activity also inhibit rosettes in the presence of antigen, but on heating the sera, a concomitant loss of reaginic activity and rosette inhibition occurs. It is proposed that there is a specific receptor on the mast cell surface for IgG₁ and reaginic immunoglobulins, and that binding to this site in the form of a complex of correct antigen ratio, will initiate the anaphylactic process.     

Allotype-Associated Differences in Concentrations of Human IgA2

Concentrations of immunoglobulin (Ig)A₂ were determined in 176 Finnish blood donor sera. Their IgA, IgM, IgE, IgG₁, IgG₂, IgG₃ and IgG₄ concentrations and Gm allotypes had been determined earlier. The mean concentration of IgA₂ was higher in individuals carrying the Gm(ax) allele (0.15 g/l) than in those negative for Gm(x) (0.103 g/l). The difference was statistically significant ( P  = 0.006). As ≥ 70% of IgA was usually IgA₁, its concentration could be calculated fairly reliably by subtracting the IgA₂ value from the IgA value. The mean IgA₁ concentration (2.03 g/l) seemed to be independent of the Gm allotypes.     

Clinical and immunological effects of immunotherapy with alum-absorbed grass allergoid in grass-pollen-induced hay fever

A double-blind, placebo-controlled study of immunotherapy was conducted in 19 patients with grass-pollen hay fever to evaluate the efficacy and safety of a formalinized depot grass allergoid. The patients were assessed before and during IT by clinical (symptom-medication scores during the grass- pollen season, specific nasal and skin reactivity) and immunological (specific IgE, IgG, IgG₁ and IgG₄ antibodies) parameters. High doses of grass allergoid, corresponding to a cumulative pre-seasonal dosage of 46050 PNU, were administered, with only one systemic reaction. The actively treated patients had significantly lower symptom-medication scores than placebo ( p < 0.01) during the month of May and showed a significant decrease in specific skin ( p < 0.01) and nasal ( p < 0.05) reactivity, and a significant early increase in specific IgE ( p < 0.01), IgG ( p < 0.0005), IgG₁ ( p < 0.001) and IgG₄ ( p < 0.05), with a subsequent decrease of IgE and IgG₁. No differences were detected in any of these parameters in the placebo group. A correlation was found between high IgG₄/IgG₁ ratio and the specific skin reactivity decrease (r = 0.691, p < 0.05), whereas a high IgG₄/IgG₁, ratio was associated with higher symptom-medication scores (r = 0.654, p < 0.05). Possible explanations of these apparent discrepancies are proposed.     

Fc Receptors in Human Placenta

Cryostat sections of placental tissue strongly adsorbed erythrocytes sensitized with IgG antibodies of human, rabbit, and guinea pig origin No adsorption occurred using erythrocytes sensitized with F(ab')₂fragments The reaction was strongly inhibited by intact IgG and by Fc fragments, weakly inhibited by pFc' fragments, and not inhibited by Facb and F(ab')₂ or albumin. These properties are similar to those of corresponding receptors in normal lymphoid tissues. Results obtained with sections of hydatidiform mole showed that the reaction occurred with the trophoblastic tissue. Porcine placenta had no Fc receptor activity. The presence of an Fc receptor in human placental tissue may therefore be of significance for the selective transfer of IgG from mother to foetus.     

Antibodies to Human Immunoglobulin Detected by Hemolysis of Human Immunoglobulin-Coated Red Blood Cells

A hemolysis assay was developed to detect alloantibodies to human immunoglobulin. A total of 1035 serum samples was tested. Anti-IgM antibodies were found in 8% of 59 normal persons and in 13% of 439 multiparous women, with the highest incidence of 67% in 341 dialysis patients. Although the anti-IgM antibodies were inhibited by both IgM and IgG, it appeared that they were also inhibited by F(ab')₂ but not by Fc. Anti-IgG antibodies were more strongly inhibited by Fc than F(ab')₂. These results suggest that anti-IgM antibodies might be analogous to antiidiotypic antibodies directed to F(ab')₂, whereas anti-IgG antibodies tend to have greater reactivity to Fc.