Immunohistochemical demonstration of FC receptors in rat tissues using immune complexes as ligand

To demonstrate the presence and localization of Fc receptors, rat liver cryostat sections were incubated with heterologous and autologous immune complexes (ICx) and immunoglobulin (Ig) aggregates. Binding was demonstrated using the immunoperoxidase technique. Autologous and heterologous ICx as well as aggregates from human and rat Ig appeared to bind to the sinusoidal wall. ICx bind in preference to aggregates. Monomeric Ig and aggregated Ig from swine and rabbit did not bind. The results demonstrated that ICx and rat and human Ig aggregates were bound via an Fc receptor. This Fc receptor was still intact in livers from carbontetra chloride and galactosamine treated rats. The receptor could also be demonstrated on spleen macrophages and on kidney interstitial cells. This method turned out to be an useful functional histochemical method to localize Fc receptors and to demonstrate their affinity and species specificity in tissues.     

The crystal structure of CHIR-AB1: a primordial avian classical Fc receptor

CHIR-AB1 is a newly identified avian immunoglobulin (Ig) receptor that includes both activating and inhibitory motifs and was therefore classified as a potentially bifunctional receptor. Recently, CHIR-AB1 was shown to bind the Fc region of chicken IgY and to induce calcium mobilization via association with the common γ-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-Å-resolution crystal structure of the CHIR-AB1 ectodomain. The receptor ectodomain consists of a single C2-type Ig domain resembling the Ig-like domains found in mammalian Fc receptors such as FcγRs and FcαRI. Unlike these receptors and other monomeric Ig superfamily members, CHIR-AB1 crystallized as a 2-fold symmetrical homodimer that bears no resemblance to variable or constant region dimers in an antibody. Analytical ultracentrifugation demonstrated that CHIR-AB1 exists as a mixture of monomers and dimers in solution, and equilibrium gel filtration revealed a 2:1 receptor/ligand binding stoichiometry. Measurement of the 1:1 CHIR-AB1/IgY interaction affinity indicates a relatively low affinity complex, but a 2:1 CHIR-AB1/IgY interaction allows an increase in apparent affinity due to avidity effects when the receptor is tethered to a surface. Taken together, these results add to the structural understanding of Fc receptors and their functional mechanisms.     

Dichotomous effect of monocyte Fc receptor interaction on anti-CD3-induced immunoglobulin synthesis

Monoclonal antibodies against the TCR/CD3 complex are capable of activating T cells which in turn may induce immunoglobulin synthesis in B cells under appropriate conditions. Here we present evidence that distinct immune responses, induced by four commonly used TCR/CD3 mAb (Leu4, OKT3, BMA030, BMA031) were related to the mAb interaction with monocyte Fc receptors for IgG. Depending on their isotype and on the technique by which they were crosslinked, TCR/CD3 mAb induced variable IgM and IgG synthesis in PBMC: If the mAb were crosslinked by monocyte IgG-Fc receptors they induced a high Ig production, while crosslinking the same mAb by plastic-bound goat anti-mouse antibodies (panning) failed to do so. Nevertheless, both crosslinking techniques triggered a strong proliferation and IL-2, IL-4, and IFN gamma lymphokine gene expression. The lack of Ig production under panning conditions was due to an additional IgG-Fc receptor interaction with monocytes: (a) If namely mAb F(ab')2 fragments, or mAb isotypes unable to bind to monocyte Fc receptors (IgG2b, IgG1 in nonresponders) were crosslinked by panning, both a good proliferation as well as Ig production ensued; (b) if TCR/CD3 mAb isotypes which could additionally bind to monocyte Fc receptor (IgG2a) were crosslinked, no Ig production occurred; (c) if mAb F(ab')2 fragments were crosslinked with a second anti-T cell antibody of IgG2a isotype, which could bind to monocyte Fc receptors, Ig synthesis was reduced. Interestingly enough, this diminishing effect, due to monocyte Fc receptor interaction, was only observed if CD4-positive cells were proliferating, but not if CD8-positive cells were activated.     

Studies on the immunoglobulin-G Fc-fragment receptor from neonatal rat small intestine.

1. A method for preparing the small-intestinal brush-border membrane of neonatal rats is described in which enzymic methods are used to remove associated polysaccharide and cell nuclei. 2. 125I-labelled IgG (immunoglobulin G) and 125I-labelled IgG Fc fragment have high specific binding and low non-specific binding to brush borders prepared in this way. F(ab)'2 fragment however, does not bind, indicating the existence of a specific receptor for the Fc fragment of IgG. The receptor system is saturable, and the affinity (KA) for the binding of rat IgG was determined by both equilibrium and kinetic methods. 3. The binding of heterologous IgG species (human and bovine) was compared and demonstrated a close similarity between human IgG and rat IgG in their receptor affinities. 4. Kinetic results are presented that are consistent with previously proposed models of ligand-induced receptor aggregation.     

An appraisal of Fc receptors on human peripheral blood B and L lymphocytes.

Human circulating lymphocytes with easily detectable surface immunoglobulin have been divided into two populations, B cells and L cells. This second population lacks membrane-incorporated Ig, but has a receptor for membrane-labile cytophilic IgG. In this study purified B and L lymphocytes were examined for Fc receptors that bind aggregated IgG and IgG complexed to erythrocytes. Purified lymphocyte populations were prepared by nylon columns and by negative selection with rosetting techniques. L lymphocytes bound aggregated guinea pig and human IgG, and formed rosettes with human erythrocytes sensitized with Ripley IgG (EA). Treatment of L lymphocytes with trypsin had no effect on the receptors for IgG. B lymphocytes did not bind EA and attachment of aggregated IgG was variable; up to one-third of these cells fixed aggregated human IgG to the cell membrane. Trypsin treatment abolished binding of Agg-IgG to B cells in sharp contrast to its effect on L cells. Furthermore, double-label immunofluorescence studies showed that cells with both membrane-incorporated Ig and receptors for aggregated guinea pig IgG were rare. These studies indicate that human peripheral blood B lymphocytes lack a high affinity, trypsin-resistant Fc receptor that is present on L lymphocytes.     

Trypanosoma musculi co-express several receptors binding rodent IgM, IgE, and IgG subclasses

The present work demonstrates the expression of receptors for the Fc portion of rodent Ig by the murine parasite Trypanosoma musculi. By using a rosette assay adapted to the parasite morphology and by flow cytometry analysis, three distinct receptors were identified. A receptor binding rabbit or rat polyclonal IgG and mouse monoclonal IgG1, IgG2a, and IgG2b was found on parasites purified from the blood and the peritoneal cavity of infected mice and on parasites maintained in culture conditions. This IgG receptor was degraded by pepsin. A separate receptor, binding only mouse monoclonal IgG3 was observed on cultured parasites. A receptor binding rabbit, rat, and mouse IgM was found on cultured and peritoneal parasites, but not on blood parasites. This receptor did not bind IgG or IgA but it bound mouse and rat IgE as well as IgM. It was degraded by trypsin. IgG and IgM/IgE receptors were co-expressed on single parasites. They were not of host origin but synthesized by trypanosomes as shown by reexpression in vitro after proteolytic degradation. Their expression was variable with the development of trypanosomes both in vitro and in vivo.     

Molecular characteristics of IgA and IgM Fc binding to the Fcalpha/muR

Fcalpha/mu receptor (Fcalpha/muR), a novel Fc receptor for IgA and IgM, is a type I transmembrane protein with an immunoglobulin (Ig)-like domain in the extracellular portion. Although IgA and IgM bind to Fcalpha/muR, the molecular and structural characteristics of the ligand-receptor interactions have been undetermined. Here, we developed twelve monoclonal antibodies (mAbs) against murine Fcalpha/muR by immunizing mice deficient in Fcalpha/muR gene. Eight mAbs totally or partially blocked IgA and IgM bindings to Fcalpha/muR. These blocking mAbs bound to a peptide derived from the Ig-like domain of murine Fcalpha/muR, which is conserved not only in human and rat Fcalpha/muR but also in polymeric Ig receptor (poly-IgR), another Fc receptor for IgA and IgM. These results suggest that IgA and IgM bind to an epitope in the conserved amino acids in the Ig-like domain of Fcalpha/muR as well as poly-IgR.     

Macrophage-T cell interactions. IV. A new receptor on T cells: Ia antigens as receptors for Ig-antigen complexes which appear early after immunization.

The Fc-receptor-positive T cells have been shown to bear a new receptor which is involved in the uptake of cytophilic complexes of Ig and antigen which are detected in the serum of mice within 6 hr following immunization. It was shown that the Fc receptor on T cells is not necessary for the uptake of these complexes. This new receptor is labile during culture of the T cells in vitro and disappears together with the Fc receptors. The new receptor is taken up by the T cells from a macrophage supernate. The evidence presented suggests that the receptor for these complexes bears determinants coded by the I region of the major histocompatibility complex but is distinct from conventional Fc receptor in that it does not bind to Fc fragments of Ig.     

Fc gamma receptors on rat eosinophils: isotype-dependent cell activation

Fc receptors for rat IgG subclasses (IgG2a, IgG2c, and IgG1) were studied on rat eosinophils by rosette formation with erythrocytes coated with monoclonal immunoglobulin (Ig) or anti-Ig antisera in a reverse assay. Inhibition experiments revealed that IgG2a and IgG2c bind to the same receptor (IgG2a/IgG2c Fc receptor), distinct from the receptor for IgG1. In addition to the recent demonstration of the blocking effect of IgG2c antibodies in immunity to schistosomes, the present results show that the existence of this common receptor led to the specific inhibition by IgG2c of IgG2a-mediated eosinophil peroxidase release. Kinetic experiments on Schistosoma mansoni-infected rat eosinophils indicate that the IgG2a/IgG2c Fc receptors were occupied by cytophilic antibodies of the IgG2a isotype during the early phase of infection and by IgG2c thereafter. By rosette experiments it was possible to displace both in vivo and in vitro cytophilically bound IgG2a from its receptor. These results confirm, therefore, the major role played by antibodies in the modulation of eosinophil effector function during schistosomiasis. They underline, moreover, the possible isotypic regulation of cell activation.     

Affinity isolation and characterization of immunoglobulin G Fc fragment-binding glycoprotein from human blood platelets.

Immune complexes bind to several eukaryotic cell types including human blood platelets through the Fc fragment of immunoglobulin (Ig) G. Utilizing immobilized Fc fragment of IgG enabled us to isolate from human blood platelets a glycoprotein of an apparent Mr = 255,000 which, upon reduction, dissociated into sub-units of an apparent Mr = 50,000. This Fc fragment-binding glycoprotein has an isoelectric point between pH 6.3 and 6.9 and is composed of 34% hydrophobic, 25% acidic, and 14% basic amino acids. The Fc fragment-binding glycoprotein was also isolated from human platelet membrane preparations and was unaffected by prior treatment of platelets with thrombin. Isolated Fc fragment-binding glycoprotein formed an in vitro complex with aggregated immunoglobulin G. These results suggest that the isolated Fc fragment-binding component may prove useful in studies concerning the functional role of glycoproteins as cellular receptors for the Fc fragment of IgG.     

Human neutrophils and eosinophils have structurally distinct Fc gamma receptors

Human Fc gamma receptors were isolated from surface radioiodinated granulocytes and eosinophils by using repetitive affinity chromatography on human IgG-Sepharose columns. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated that cell preparations containing eosinophils possessed a 43,000 Mr Fc gamma-binding macromolecule. Nylon wool-filtered cells from patients with eosinophilia and cell cultures derived from normal donors provided highly purified eosinophil preparations that expressed only the 43,000 Mr Fc gamma receptor. Granulocyte populations yielded the 52,000 to 68,000 Mr Fc gamma receptor characteristic of neutrophils as well as the Fc gamma-binding macromolecules apparently derived from eosinophils. The 43,000 Mr Fc gamma receptor of the eosinophil and the 31,000 and 34,000 Mr fragments that appear to be derived from it were able to rebind selectively to human IgG1-Sepharose, Fc gamma 1-Sepharose, IgG3-Sepharose, and Fc gamma 3-Sepharose. In contrast, the 52,000 to 68,000 Mr Fc gamma receptor from neutrophils could rebind only to IgG1-Sepharose and Fc gamma 1-Sepharose. The results demonstrate that the Fc gamma receptor of human eosinophils is distinct in structure from the neutrophil Fc gamma receptor and that these Fc gamma receptors, at least in their solubilized states, differ in specificity for human IgG3.     

Lipopolysaccharide and interleukin 1 inhibit interferon-gamma-induced Fc receptor expression on human monocytes

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.     

Recombinant soluble Fc epsilon receptor II (Fc epsilon RII/CD23) has IgE binding activity but no B cell growth promoting activity

We have undertaken the production of recombinant soluble Fc epsilon receptor II (Fc epsilon RII) as a secretory protein, but not as a cleavage product of membrane-bound receptor. Several plasmid constructs containing soluble receptor sequence were prepared. Only a chimeric gene containing the sequences encoding IL-6 signal peptide and the soluble moiety of Fc epsilon RII could be expressed in Xenopus laevis oocytes and CHO cells, resulting in the secretion of soluble Fc epsilon RII. The recombinant soluble Fc epsilon RII was also produced in the yeast expression system. The NH2-terminal sequence analysis of the chimeric gene product generated by oocytes demonstrated the correct cleavage of IL-6 leader sequence by a signal peptidase. Moreover, most of CHO cell and all of the yeast-derived recombinant molecules were products identical with the native B cell-derived soluble Fc epsilon RII. These recombinant products as well as the natural soluble receptor derived from a human B cell line could bind both human IgE and two different anti-Fc epsilon RII mAb and could competitively inhibit the binding of IgE to Fc epsilon RII-expressing cells. However, the recombinant soluble Fc epsilon RII and highly purified native molecules did not display any B cell growth-promoting activity.     

A two-step selection approach for the identification of ligand-binding determinants in cytokine receptors

We have developed a novel cell-based method for the isolation and selection of mutant cytokine receptors with defects in ligand binding and applied it to the human interleukin-4 receptor. The experimental procedure is based upon the functional heterologous expression of receptor mutants in eukaryotic cells followed by a two-step selection procedure. Positive selection for cells that express receptor variants is achieved by means of an agonistic antibody that mediates cell survival through receptor dimerization. An IL-4-coupled toxin is subsequently used to select against cells expressing wild-type receptors. Cells expressing mutant receptors that are unable to bind the cytotoxic ligand survive and can be amplified. The procedure allows the isolation of rare receptor variants from cell pools containing predominantly wild-type cells. This method, which should be equally applicable to similar receptor systems, was used to demonstrate the importance of a critical charged amino acid residue in the human IL-4 receptor alpha-subunit for IL-4-induced receptor activation.     

Streptococcal Fc receptors. II. Comparison of the reactivity of a receptor from a group C streptococcus with staphylococcal protein A

The reactivity of a soluble Fc receptor from a group C streptococcus ( FcRc ) was compared antigenically and functionally with the staphylococcal Fc receptor, protein A. Protein A and FcRc were found to inhibit each others' binding to the Fc region of human IgG, indicating that they bind to sites that are in close proximity on the Fc region of human IgG. The two bacterial Fc receptors were antigenically unrelated. Differences were observed in the species and subclass reactivity of the two receptors. The patterns of binding of protein A and FcRc under various conditions suggested that these receptors reacted with distinct regions on the Fc region of immunoglobulins. FcRc bound more efficiently to goat, sheep, and cow IgG, protein A bound more efficiently to dog IgG, and neither receptor bound to rat IgG. Differences were also observed in the reactivity towards human IgG subclasses. The FcRc bound to all samples of the four human IgG subclass standards. Protein A bound to IgG1, IgG2, and IgG4, and to one of two IgG3 myeloma proteins tested. The reactivity of our soluble FcRc corresponds to a type III streptococcal Fc receptor classified by the reactivity of intact bacteria.     

Receptors for IgM on certain human B lymphocytes.

A receptor for IgM was demonstrated on the surface of human B lymphocytes by using a rosette technique with ox erythrocytes coated with rabbit IgM antibody (EAM). Lymphocytes forming rosettes with EAM did not bind sheep red cells, had membrane Ia-like antigens and, in some instances, surface immunoglobulin. The specificity of EAM rosettes was confirmed by inhibition experiments with purified human Ig. IgM but not IgG molecules inhibited the rosette reaction. In addition, inhibition of EAM rosettes with IgM fragments showed that the receptor has affinity for a part of the molecule located in the Fc portion. By analogy with the receptors previously found on certain human T cells, receptors for IgM were not detected on freshly isolated B cells, but were expressed after overnight culture in IgM-free media. Studies on different human lymphoid tissues showed that IgM receptors are expressed on a limited percentage of both circulating and noncirculating B cells. In addition to normal B cells, the malignant B cells of a majority of cases of chronic lymphocytic leukemia expressed the receptors for IgM.     

Recombinant human IgG1-murine IgE chimeric Ig. Construction, expression, and binding to human Fc gamma receptors

We have constructed a set of chimeric Ig by exchanging corresponding H chain C domains between human (hu) IgG1 and murine (m) IgE. We used this set of Ig to dissect the interaction of individual Ig domains with human Fc gamma receptors. Only one of the chimeras, epsilon/C gamma 2,3 (an mIgE with C epsilon 3 and C epsilon 4 replaced by C gamma 2 and C gamma 3 from huIgG1), binds tightly to the human Fc gamma RI on U937 cells. We found that epsilon/C gamma 2,3 has only twofold lower affinity for Fc gamma RI as compared to huIgG1. The gamma/C epsilon 4 (huIgG1 with C epsilon 4 replacing C gamma 3) binds weakly to Fc gamma RI. The other chimeric Ig, epsilon/C gamma 3, epsilon/C gamma 2, and gamma/C epsilon 3, as well as mIgE do not bind detectably to Fc gamma RI. From these data we conclude that the C gamma 2 domain is crucial for binding and contains the majority of the binding site for Fc gamma RI on IgG1. The C gamma 3 domain makes a smaller contribution to the binding, and the C gamma 1 domain and the hinge region have very little effect on the Fc gamma RI-IgG1 interaction. The chimeric epsilon/C gamma 2,3 and huIgG1 both mediate the formation of rosettes between K562 cells and antigen-sensitized E with similar concentration dependences. These results suggest similar ability to bind to Fc gamma RII. The other chimeric Ig do not cause rosettes in this assay system. Hence, both C gamma 2 and C gamma 3 seem to be required for binding to Fc gamma RII, but the C gamma 1-hinge region has no detectable effect.     

X-ray crystallographic studies of IgG-Fc gamma receptor interactions

Human Fc gamma receptors (Fc gamma Rs) for the Fc portion of IgG are major mediators of the adaptive immune response. Crystal structures of their extracellular domains were recently solved and shown to obey a common fold, resulting in a heart-shaped structure. Together with the multifunctional Fc fragment, whose structure was deciphered in the 1970s, the complex of an Fc gamma R with Fc was recently crystallized. Its crystal structure indicates that despite the dimeric character of the Fc fragment, only one Fc gamma R can bind to Fc, due to an introduced asymmetry within the homodimeric Fc, as well as by binding to symmetrically related residues of both Fc chains. Homology modelling suggested that the other Fc gamma Rs bind the Fc in a similar manner. The resolved complex structure can be regarded as a paradigm for Ig binding to Fc receptors and serves as a solid base for the design of compounds interfering with complex formation. Such molecules would be of invaluable benefit for the therapy of immunological disorders.     

Fc gamma RI and Fc gamma RII on monocytes and granulocytes are cytotoxic trigger molecules for tumor cells

As part of an effort to define the cytotoxic trigger molecules on human myeloid cells, the ability of the different Fc receptors for IgG (Fc gamma R) to mediate killing of tumor cell lines by monocytes and granulocytes was examined. This was accomplished by studying cytolysis of hybridoma cell (HC) targets bearing surface antibody directed toward the different Fc gamma R. The HC line, HC IV.3A, which bears Ig directed to the low affinity Fc gamma R (Fc gamma RII) on monocytes and neutrophils was lysed by human monocytes. The extent of lysis of HC IV.3A was approximately equal to that of anti-Fc gamma RI (the high affinity Fc gamma R on human monocytes) bearing HC lines (HC 32.2A and HC 62A) and was not augmented by treatment of the monocytes with interferon-gamma (IFN-gamma). In contrast, neutrophils lysed HC IV.3A and HC 32.2A only after activation with IFN-gamma. Since Fc gamma RI is not detectable on untreated neutrophils and is induced by IFN-gamma on these cells, lysis of HC 32.2A by IFN-gamma-activated neutrophils correlated with receptor induction. On the other hand, Fc gamma RII was present at equal levels on untreated and IFN-gamma-treated neutrophils, but only IFN-gamma-treated neutrophils mediated cytotoxicity via Fc gamma RII. In this case, enhanced killing appeared to be due to events other than an increase in Fc gamma RII number. Neither untreated nor IFN-gamma-treated neutrophils mediated the lysis of the anti-Fc gamma RIII bearing HC 3G8A. Thus, binding to the tumor target via this Fc receptor does not lead to lysis and may initiate signals distinct from those triggered through Fc gamma RI or Fc gamma RII. Surprisingly, HC bearing high amounts of mouse IgG1 antibody of irrelevant specificity were also lysed by monocytes. This lysis was blocked by soluble IV.3 antibody and thus appeared to be due to binding of the Fc portion of the surface Ig to Fc gamma RII on monocytes. Furthermore, monocytes from donors with a form of Fc gamma RII incapable of binding aggregated mouse IgG1 did not lyse these HC, but displayed normal lysis of HC IV.3, demonstrating that this structurally different Fc gamma RII remained a functional trigger molecule. Overall, these studies have demonstrated the specificity of Fc receptors in triggering monocyte- and granulocyte-mediated antibody-dependent tumor cell killing and have begun to dissect functional similarities and differences among the three defined Fc gamma R on human myeloid cells.     

The immune response in the hamster. VII. Studies on cytophilic immunoglobulin.

Hamster 7S IgG1 and IgG2 antibodies to hen-egg albumin (HEA) were tested for their capacity to bind to macrophage cytophilic Ig receptors. Both IgG1 and IgG2 were cytophilic for hamster macrophages though the membrane receptor had a predominant specificity for IgG1. Hamster IgG1 bound primarily to homologous macrophages whereas IgG2 bound to macrophages from other rodent species as well. The binding of hamster Ig to hamster macrophages was inhibited by a wide range of heterologous rodent sera. The only exception was guinea pig serum since guinea pig IgG2 was found to bind only to homologous macrophages. Sheep red blood cells (SRBC) coated with hamster IgG2 were ingested by macrophages more readily than those coated with hamster IgG1. Thus, there appeared to be a paradoxical relationship between the apparently strong affinity of IgG1 for the hamster macrophage Ig receptor and its reactivity weak ingestion promoting activity. Implications of this phenomenon are discussed.