Subclass Specificity Research Articles

Pharmacological classification of benzamides.

The problem of classifying benzamides is, in general, the same as classifying neuroleptics. A pharmacological classification of neuroleptics can be established on the basis of the following criteria: specificity of action on dopaminergic receptors; penetration into the CNS; dopaminergic profile in the CNS; relative binding with respect to subclasses of DA; preferential antagonism of certain effects of apomorphine; preferential affinity for dopaminergic structures; activating effects on DA systems at low doses; different clinical effects: antiemetic, psychiatric (antihallucinatory and disinhibitory), neurologic. A relationship between biochemical, behavioural and clinical effects is proposed: antihallucinatory effect: decrease of dopaminergic function; disinhibitory effect: increase of dopaminergic function. We emphasize the problem of doses used because the different effects occurred for a given drug at different doses. This hypothesis suggests that positive symptomatology of schizophrenia is related to hyperdopaminergic activity, negative symptomatology is related to hypodopaminergic activity. Classification of benzamides: metoclopramide: peripheral dopaminolytic activity antiemesis without psychiatric or neuroleptic effects with low doses. sulpiride, tiapride, DAN 2163: peripheral effects + preferential blockade of D3 and D4 receptors, central DA activation with low doses, disinhibitory effects. With high doses, decrease of specificity for subclasses of DA receptors, central DA inhibition, antihallucinatory effects but little extrapyramidal symptomatology and sedation. sultopride: central DA inhibition, antihallucinatory effects, sedative and neurological effects.

Isotypic and allotypic specificity of mouse rheumatoid factors.

The specificity of polyclonal mouse rheumatoid factors (RF) was analyzed by competition experiments with heat-aggregated mouse IgG subclasses. The RF spontaneously produced by three normal mouse strains (129/Sv, CBA/Ht, and C57Bl/6) and by two strains with autoimmune diseases (MRL/l and NZB) were found to consist of distinct non-cross-reactive antibody subpopulations each specific for one IgG subclass. The sera of the normal strains contained IgG1- and IgG2a-specific RF. The autoimmune strains produced an additional variety of RF that was specific for The autoimmune strains produced an additional variety of RF that was specific for IgG2b. Also, the RF secreted by spleen cells of various normal strains after in vitro polyclonal activation with lipopolysaccharide could be resolved into distinct subpopulations specific for IgG1 or IgG2a. These results were confirmed by the analysis of monoclonal RF derived from BALB/c, C57Bl/6, CBA/Ht, and 129/Sv mice: of 73 hybridomas with RF activity, 71 displayed a strict subclass specificity. The subclass predominantly recognized depended on the origin of the spleen cells used to generate the hybridomas. After polyclonal activation in vitro, a broad spectrum of different specificities was obtained with 16 RF specific for IgG1, 13 for IgG2a, and 4 for IgG2b. In contrast, 27 of 28 monoclonal RF derived from 129/Sv and BALB/c mice without prior polyclonal activation were specific for IgG2a, and of these 75% were allotype specific since they failed to react with IgG2a of the b allotype. These results demonstrate the importance of subclass specificity in the production of RF in vivo. With the exception of the IgG2b-specific clones, all these monoclonal RF reacted preferentially with heat-aggregated or antigen-bound IgG. Among the hybridomas generated by the fusion of in vitro polyclonally activated spleen cells of 4-wk-old mice, the frequency of clones with RF activity was at least 40 times higher than that of clones specific for mouse IgM, human IgG, ovalbumin, and hen lysozyme.

Monoclonal antibody specific for avian sarcoma virus structural protein p27.

A hybridoma cell line which secretes antibody to the Rous sarcoma virus (RSV) structural protein p27 has been established. The hybrid resulted from the fusion of NS-1 myeloma cells with spleen cells from a Balb/c mouse which was immunized with RSV-transformed mouse cells. Antibodies produced by the hybrid clone immunoprecipitated p27 and gag precursor proteins (Pr180gag,pol, Pr76gag and Pr66gag) from [35S]methionine-labelled chicken embryo fibroblasts transformed by the Schmidt-Ruppin strain of RSV. When Schmidt-Ruppin virus was radioactively labelled with [35S]methionine, p27 was the only virus structural protein immunoprecipitated. Antibody production by the hybrid clone (designated 7-29-D6) has remained stable for longer than 12 months at a level of 50 micrograms IgG/ml medium. A highly sensitive method to determine the subclass specificity of monoclonal antibodies is described. In this procedure, the clone is incubated with [35S]-methionine, and radiolabelled antibody is precipitated with affinity-purified, subclass-specific rabbit anti-mouse serum and Staphylococcus aureus. The advantages of this procedure are discussed.

Plasma exchange in the treatment of hemolytic disease of the newborn.

Plasma exchange has become a widely used therapeutic tool in the treatment of immune-mediated disease. This technique has been applied to the treatment of gravid women whose high antibody titers and past history of stillbirths indicate a significant possibility of fetal loss due to HDN. The procedure was effective in decreasing both antibody titer and the quantity of antibody in four of five patients. In one other case, the antibody titer rose toward the end of the pregnancy; however, the quantity of anti-D remained low. The IgG:IgM ratio and the subclass specificity of IgG did not significantly change with treatment. Hematologic monitoring also indicated no adverse effects, and three of the five women delivered viable fetuses. Two of the other women produced infants only moderately affected by HDN but stillborn within days of intrauterine transfusion. The results indicate that plasma exchange is a useful therapeutic consideration in the sensitized gravid women.

Specific binding of bovine, ovine, caprine and equine IgG subclasses to defined types of immunoglobulin receptors in gram-positive cocci

One hundred and twenty bacterial strains were tested for non-immune binding of radiolabelled bovine, ovine, caprine and equine immunoglobulins. Bacteria possessing previously defined IgG receptors interacted in a well defined manner with purified IgG subclass immunoglobulins. Human group C and G streptococci carrying IgG receptors type III were capable of binding all IgG subclasses in the four mammalian species studied. Protein A-containing staphylococci demonstrated a restricted specificity with binding of bovine IgG1, ovine IgG1, caprine IgG1 and IgG2 as well as equine IgG(ab). Group A streptococci which can bind human IgG did not show specific reactivity. A new type of binding unrelated to the regular Fc-mediated binding was observed with equine IgG(T). The differences in specificity for IgG subclasses suggest that structures with binding capacity to streptococcal type III Fc receptors are different from staphylococcal protein A reactive sites. Inhibition experiments performed with purified immunoglobulins showed that individual IgG subclasses differed greatly in their inhibiting capacity reflecting differences in avidity. The high avidity and the broad, unrestricted immunoglobulin G reactivity of streptococcal IgG receptor type III indicate that human group C and G streptococci may provide a valuable tool for solid phase absorption of immunoglobulins from several mammalian species.

Purificaton of a functional mouse Fc receptor through the use of a monoclonal antibody.

We recently reported the isolation of a rat monoclonal antibody designated 2.4G2 (9) that is directed against the mouse trypsin-resistant Fc receptor (FcR) for IgG2b and IgG1 immune aggregates. We have now utilized the Fab fragment of 2.4G2 as an affinity reagent to purify FcR from the macrophage cell line J774 to apparent homogeneity. The antigen isolated from J774 cells consisted of two general types of polypeptides with broad electrophoretic mobilities of approximately 60,000 and 47,000 mol wt. Similar broad bands ranging from 47,000 to 70,000 mol wt were isolated from various FcR-bearing cell lines of B, T, and null lymphocyte, as well as of macrophage origin. J774 FcR was judged to be a glycoprotein based on the sensitivity of its isoelectric point to neuraminidase digestion, its labeling with galactose oxidase/NaB[3H4], and its binding to concanavalin A-Sepharose. In phosphate-buffered saline, the isolated protein formed large aggregates that were shown to retain FcR activity, albeit with a somewhat altered IgG subclass specificity. The FcR aglutinated erythrocytes that were coated with both IgG2b and IgG2a that did not otherwise hemagglutinate. In addition, iodinated FcR bound to Sephadex beads coated with rabbit IgG, mouse IgG1, IgG2b, and IgG2a, but not to beads coated with mouse IgG3 or rabbit F(ab')2 fragments. The binding of the purified receptor to all IgG classes was inhibited by the Fab fragments of 2.4G2. In contrast, the binding of IgG2a to intact macrophages was inhibited by 2.4G2 Fab by only 15%, whereas rabbit IgG immune aggregate binding was almost completely abolished.

Demonstration of a new type of immunoglobulin G receptor in Streptococcus zooepidemicus strains.

Forty-seven bacterial strains representing four different group C streptococcal species were tested for binding of human and bovine immunoglobulin G (IgG) subclasses. Specific binding sites for IgG were found in all bacterial species studied. The four species included differed, however, in their capacities to interact with various IgG subclasses, indicating the existence of different types of IgG receptors. Streptococcus equisimilis and Streptococcus dysgalactiae were found to carry the same type of IgG receptor, one that is identical to the previously described Fc-binding structure type III. A new type of bacterial IgG receptor was detected in Streptococcus zooepidemicus strains. This receptor exhibits a protein A-like subclass specificity, with binding of human IgG1, IgG2, and IgG4 and of bovine IgG2. However, differences in their capacities to interact with other non-human immunoglobulins indicated that these two immunoglobulin-reactive structures were different. All types of IgG receptors in group C streptococci were found to be heat stable but susceptible to proteolytic enzymes. The inability of human serum albumin or fibrinogen to inhibit the uptake of radiolabeled IgG shows that the IgG receptor is separate from binding sites for these two other proteins on the bacterial cell surface. The existence of similar IgG receptors in closely related streptococcal species suggests that these structures have a common origin.

The Characterization and Functional Significance of Plasma Membrane fc Receptors

The importance of various components of the plasma membrane in the generation of the immune response has long been recognized. The identification and characterization of functional membrane receptors are essential for an understanding of the molecular basis of the immune response. The Fc receptor can be operationally defined as a site on the plasma membrane which is capable of binding the Fc portion of IgG. The Fc receptor has been reported on both lymphoid and nonlymphoid cell types. The functional significance of the receptor on these cell populations, as well as the biochemistry of the receptor, is not well established and is currently an important area of investigation. In this review, the Fc receptor will be discussed, primarily in human and murine lymphoid and macrophage cell populations, as a model of membrane immunoreceptors. The review will be divided into four sections. In the first section, consideration will be given to the binding assays used to study Fc receptor-ligand interactions and the class and subclass specificity of the receptor. The possibility of receptor-receptor interaction and quantitation of receptor sites will be discussed. The second phase of the review will be concerned with the fractionation and biochemistry of this receptor system, emphasizing the differences reported in receptor composition from different cell sources. In the third section of the review, the biologic significance of the receptor will be examined. The importance of the Fc receptor in opsonization and phagocytosis by mononuclear phagogytes, B-cell activation, antibody-dependent cell cytotoxicity, and the possible relationship of Fc receptors to the histocompatibility-linked immune response genes will be discussed. Finally, in the fourth section, the possible mechanisms of receptor regulation will be considered. Cell fusion experiments which have been performed and the expression of receptor activity on the hybrid cell clones will be discussed. Receptor regulation at the physiologic level will include the effects of cyclic nucleotide levels and cell cycle on receptor expression. It is the intent of this review to provide a detailed analysis of the Fc receptor and to emphasize its importance in microbiology, cell biology, and immunology.

The Subclass Specificity for the Binding of Murine Myeloma Proteins to Macrophage and Lymphocyte Cell Lines and to Normal Spleen Cells

Abstract Murine myeloma proteins of the four IgG subclasses and of the IgA and IgM classes have been examined for their ability to inhibit the binding of affinity cross-linked trimeric rabbit IgG to various types of murine cells. In addition, proteins of the IgG1, IgG2a, and IgG2b subclasses have been cross-linked with dimethyl suberimidate and the purified oligomers have been tested for direct binding. With a macrophage line (P388D1), a lymphocyte line (AKTB-1), and normal spleen cells, proteins of the IgG1, IgG2a, and IgG2b subclasses all compete for binding at the same site. Differences in binding affinities between proteins of the IgG1, IgG2a, and IgG2b subclasses are small and are not greater than differences in affinities when proteins within a subclass are compared. Proteins of the IgG3 subclass and the IgM and IgA classes do not significantly inhibit IgG binding at the highest concentrations tested. P388D1 cells are also able to bind iodinated monomeric IgG2a proteins with higher affinity than expected from inhibition studies. This binding is inhibitable by proteins of the IgG1, 2a, and 2b subclasses, and is trypsin sensitive. These results could be explained by the existence of a second type of receptor, or by assuming that the iodination procedure increases the affinity of IgG2a proteins for P388D1 cells.

Binding Site of Human IgG Subclasses and Their Domains for Fc Receptors of Activated Murine T Cells

Abstract A subpopulation of activated murine T cells (25%) were found to bear Fc receptors for mouse, rabbit, and human IgG. Studies, by rosette inhibition and indirect immunofluorescence, of their specificity for human IgG subclasses have shown that these receptors bound nonaggregated IgG1, IgG2, and IgG3 in a comparable fashion, but did not bind IgG4. The binding site has been localized exclusively to the Fc region and the binding capacity of this fragment was not affected by mild reduction. Binding of the Fc fragment of IgG4 could not be demonstrated. Using isolated Cγ2 and Cγ3 (pFc′) domains from IgG1 and a Cγ3-derived fragment (Fc′), a major binding site was localized to the Cγ3 domain between residues Gln342 and His433. A 10-fold molar excess of Cγ3 dimer over native Fc was, however, required to show comparable activity. The monomeric Cγ2 domain was 10-fold less active than the Cγ3 fragment. Human urine β2-microglobulin was not cytophilic. These data are discussed and it is suggested that the Cγ2 and Cγ3 domains may contribute to the formation of a cooperative binding site through quaternary interdomain interactions. By redistribution experiments, it was demonstrated that the three human IgG subclasses shown to be cytophilic share common or linked receptors, which were not associated with the H-2 alloantigens.

Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line.

To evaluate subclass specificity and aggregate size requirements of IgG receptors on mouse cells, we measured binding of radiolabeled monomeric and BDB-aggregated mouse myeloma proteins fractionated into various sizes by means of gel filtration. Monomers, tetramers, and high molecular weight (approximately 10(7) daltons) aggregates were used. The various cells and cell lines studied could be segregated into three patterns of reactivity: (a) Macrophage and macrophage-like cell lines bound monomer IgG2a preferentially; high molecular weight IgG aggregates bound as follows: IgG1 = IgG2b = IgG2a. (b) Lymphoid lines D2N and S49 showed no capacity to bind monomer IgG2a; high molecular weight aggregates bound as follows: IgG1 = IgG2b less than IgG2a. (c) Other Thy-1-positive lymphoid cell lines (EL4 and L5178) and normal T and B cells showed no capacity to bind monomer IgG; high molecular weight IgG aggregates bound to a lesser extent than to cells of the first two categories in the following manner: IgG1 less than IgG2b greater than or equal to IgG2a. The variable pattern of reactivity of the macrophage-like cell lines with monomer and aggregated IgG suggested that two distinct receptors for IgG were present: one capable of binding IgG2a and another capable of binding all aggregates. Further evidence for this hypothesis was obtained by analysis of the inhibitory capacity of different IgG subclasses on the binding of aggregated IgG and monomer IgG2a to P388 cells. Inhibition of monomer IgG2a binding was effected only by monomer or aggregated IgG2a, whereas inhibition of binding of aggregated IgG1 or IgG2b was noted with aggregates of all three subclasses with some preferential inhibition by monomer IgG2b being observed. Furthermore, monomer IgG2b binding was preferentially inhibitable by monomer IgG2b. It is postulated from these data that two receptor sites are present on this macrophage-like cell line, one reactive with aggregates of all three subclasses as well as monomer IgG2b, and another receptor specific for monomer IgG2a which also binds aggregated IgG2a. Support of this concept was obtained by trypsinization experiments in which the binding of monomer IgG2a was markedly decreased by trypsin treatment of cells, whereas the binding of aggregated IgG2b was unaffected by this treatment.

Lactate dehydrogenase (LDH)-IgG 3 immunoglobulin complexes in human serum

Sera of fifteen patients containing complexes of LDH and IgG were analysed. The LDH-IgG complexes of fourteen of these patients were investigated for their subclass specificity. They appeared to consist of LDH and IgG 3, except for one of the patients. The serum of the latter patient also contained complexes of LDH and IgG 1. In this serum both types of the immunoglobulin light chains were involved in the formation of the complexes, whereas in the serum of the patients with LDH-IgG 3 complexes alone, they only contained one of the two immunoglobulin light chain types; it mainly concerned kappa type. The results of the present study were compared to those of a previous study on LDH-IgA complexes, in which the IgA fraction always contained light chains of kappa type. The mean LDH activity and the heat stability of the LDH activity in the sera with LDH-IgA complexes appeared to be higher than in the sera with LDH-IgG 3 complexes. In contrast to complexes of LDH and IgA which occurred in all age groups LDH-IgG 3 complexes were mainly found in elderly persons. Although auto-antibodies to different tissue antigens were present in the majority of the sera containing LDH-IgG 3 complexes, antibodies to LDH could not be demonstrated. A relationship between the presence of the complexes in the serum and a particular disease could not be established.

Cellular Differentiation in a Murine Myelomonocytic Leukemia

Abstract Mouse peritoneal macrophages and macrophages derived from normal bone marrow progenitors in vitro possess surface receptors whose predominant specificity is directed toward the IgG2a subclass of IgG moleclues. Myelomonocytic leukemic cells also possess such IgG receptors but in an abnormally low frequency and without demonstrable subclass specificity. Within the leukemic population, monoblasts are distinguished from myeloblasts by their greater adhesiveness to glass, higher frequency of cells with surface receptors for IgG, and by erythrophagocytosis. Leukemic monoblasts, myeloblasts, and their morphologically mature progeny demonstrate impaired phagocytic ability. A concept of the development of surface receptors for IgG in the monocyte-macrophage cell line is presented.

IgG subclass specificity of human monocyte receptor sites.

MONOCYTES as well as macrophages have receptor activity for IgG and the third component of complement1–5. The receptor sites may act independently or cooperatively in binding and ingestion of red cell-antibody complexes2–5. Myeloma proteins of known subclass specificity used as inhibitors of phagocytosis of various red cell–IgG antibody complexes provided suggestive evidence for a subclass specificity of these receptor sites1. We wish to report experiments which define this subclass specificity further and indicate similarities for the binding sites of both γG1 and γG3.

The interaction between human monocytes and red cells. Specificity for IgG subclasses and IgG fragments.

Studies were carried out in order to characterize the specificity of IgG sub-classes and IgG fragments for rosette formation using red cells and human mononuclear cells. Rosette formation of red cells coated with anti-D was inhibited by free IgG(1) and IgG(3); less inhibition occurred with IgG(2) and IgG(4). Red cells specifically coated with IgG(1) and IgG(3) by chromic chloride were bound to monocytes. Rosette inhibition of anti-D-coated red cells occurred with free Fc fragment of IgG globulin, and only partly with F(ab')(2). Inhibitory capacity of Fc fragments of IgG and gamma(1) heavy chain from heavy chain disease was reversed by the cleavage of disulfide bonds. No inhibition was noted with Fab, or with pepsin components II, III, or IV. These studies indicated that the mononuclear receptor was specific for IgG(1) and IgG(3). The peptide portion of IgG globulin which attached to the mononuclear cell appeared to reside in the N-terminal portion of the Fc fragment and also appeared to require the integrity of the inter-heavy chain disulfide bond. A specific receptor for C3 was not confirmed.

Subclasses of human immunoglobulin a based on differences in the alpha polypeptide chains.

Antiserum from goats immunized with heavy polypeptide chains from a gammaA-type myeloma globulin was absorbed with serum from patients with selective absence of immunoglobulin A (gammaA). The resulting reagents could be used for the classification of 58 gammaA-myeloma proteins into two distinct antigenic types, respectively called subclasses He and Le. These differences were shown to be related to the heavy (alpha) polypeptide chains and independent of the integrity of interchain disulfide bridges. The gammaA-immunoglobulin from normal serum appears to consist, for the most part, of molecules with Le subclass specificity.