Structural basis of the interaction between IgG and Fcgamma receptors.

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.

Interleukin 17, a T-cell-derived cytokine, promotes tumorigenicity of human cervical tumors in nude mice.

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.

Identification of the cleavage site involved in production of plasma soluble Fc gamma receptor type III (CD16).

CD16 (FcgammaR type III) is a low-affinity IgG Fc receptor (R) that exists in two isoforms, a transmembrane FcgammaRIIIa expressed by NK cells and monocytes, and a phosphatidylinositol-linked FcgammaRIIIb expressed by neutrophils. A soluble form of CD16 (sCD16) circulates in plasma. The cleavage site and the nature of the enzyme(s) involved in production of sCD16 were investigated. Soluble CD16 was purified to apparent homogeneity from human serum by eight steps, including anion exchange and immunoaffinity chromatography. Serum sCD16 was sequenced at both ends, as well as a recombinant form of sCD16 used as control. N-terminal sequencing demonstrated that serum sCD16 originates from neutrophil FcgammaRIIIb and C-terminal sequencing suggested that the cleavage site is between Val 196 and Ser 197, close to the membrane anchor. Addition of a hydroxamate-based inhibitor of Zn2+ metalloproteinases (RU36156) led to a dramatic decrease of sCD16 production by phorbol 12-myristate 13-acetate-activated neutrophils, whereas inhibitors of serine proteinases had no significant effect, showing the metalloproteinase dependence of this cleavage process.

N-glycan structures of a recombinant mouse soluble Fcgamma receptor II.

N-glycans of a recombinant mouse soluble Fcgamma receptor II (sFcgammaRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226:139-46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was alpha-(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and alpha-(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry. Carbohydrate sequence [see text]

Affinity of the interaction between Fc gamma receptor type III (Fc gammaRIII) and monomeric human IgG subclasses. Role of Fc gammaRIII glycosylation.

Binding of the Fc region of IgG antibodies to low affinity Fc gamma receptors (Fc gammaR) triggers important effector functions in the immune system. The type IIIb Fc gammaR (Fc gammaRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of Fc gammaRIIIb (sFc gammaRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab')2) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K(A)) of 1.3 +/- 0.6 x 10(6) M(-1) and 2.6 +/- 0.4 x 10(5) M(-1), respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 +/- 0.2 x 10(6) M(-1)), whereas that for human IgG3 was twofold higher (4.2 +/- 0.4 x 10(5) M(-1)). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 >> IgG4 >>> IgG2. Thus, the extracellular polypeptide of Fc gammaRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.

Soluble Fc gamma receptors: interaction with ligands and biological consequences.

Soluble Fc gamma receptors are produced by cleavage of the membrane receptors or by alternative splicing. They are found in biologic fluids. After a brief description of the structure and mode of production of soluble Fc gamma R, we address the question of ligands and function of the soluble Fc gamma R by using recombinant molecules and transgenic animals. We show that soluble Fc gamma R are not only IgG-binding factors which interfere with, and block, Fc-dependent immune reactions but also molecules that interact, in vitro, with non-Ig-ligands such as CR3 and CR4 and are trigger or regulate immune functions via these receptors.

A transforming growth factor beta-like immunosuppressive factor in immunoglobulin G-binding factor.

Immunoglobulin G-binding factors (IgG-BF), which are produced by cells of the immune system, inhibit antibody production. In this paper, we show that transforming growth factor-beta (TGF-beta) suppresses secondary in vitro anti-sheep red blood cell responses of mouse splenocytes and lipopolysaccharide- or anti-IgM-stimulated mouse B cell responses in a way similar to, and with the same kinetics as, rodent IgG-BF. Moreover, the immunosuppressive activity of IgG-BF was totally neutralized by polyclonal and monoclonal anti-TGF-beta antibodies and it eluted with TGF-beta by gel exclusion chromatography, suggesting that a TGF-beta-like immunosuppressive factor is present in IgG-BF. We also show that TGF-beta behaves as an IgG-BF since it binds to insolubilized IgG, but not to insolubilized F(ab')2 or bovine serum albumin. Altogether, the data support the concept of a biological role for TGF-beta in the IgG-mediated negative feedback of antibody responses.

Recombinant soluble Fc gamma receptors: production, purification and biological activities.

Soluble forms of low affinity receptors for the Fc portion of IgG circulate in body fluids and regulate immune functions. We describe the transfection, production and purification techniques which allow the preparation, at a laboratory scale, of milligram amounts of glycosylated recombinant mouse and human soluble Fc gamma receptors. These recombinant products bind IgG and are biologically active on immune responses, like their normal counterparts.

Natural and recombinant soluble low-affinity Fc gamma R: detection, purification, and functional activities.

Studies on the identification, cloning, and biochemical characterization of natural and recombinant human and mouse low-affinity soluble Fc gamma R (sFc gamma R) have been developed using various methods. RT-PCR and/or biochemical analyses have demonstrated that low-affinity sFc gamma R (i) are generated by enzymatic cleavage of membrane-associated receptors or by an alternative splicing of the transmembrane region encoding exon and (ii) comprise only the extracellular domains or these domains plus the intracellular region of the membrane-associated molecules, respectively. Functional studies indicated that recombinant sFc gamma R bind mouse and human IgG subclasses with a binding profile identical to that of their membrane counterparts and inhibit Fc gamma R-mediated functions such as immune complex binding or ADCC. In addition, it has been demonstrated that a mouse recombinant truncated sFc gamma RII inhibits antibody responses to T-dependent antigens as well as B-cell proliferation and that a human recombinant truncated sFc gamma RIIIB blocks the Ig production by activated human peripheral blood mononuclear cells. Finally, different immunoassays devised to detect and quantitate circulating sFc gamma R showed that sFc gamma R serum levels vary in circumstances such as injections of protein antigens, in parasitic infections, in tumor-bearing mice, in patients with multiple myeloma (MM), or upon infusions of IgG or Fc gamma fragments in MM or immune thrombocytopenic purpura patients. The use of recombinant sFc gamma R, as well as the availability of monoclonal and polyclonal antibodies directed against different regions of these molecules, makes it possible to characterize further the biological effects of sFc gamma R and their biochemical and immunochemical characteristics, as well as to define their putative ligands on cell membranes.

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc gamma R) encoded by an alternatively spliced transcript of the Fc gamma RII gene.

Low affinity Fc gamma R are a heterogeneous group of glycoproteins which exist in transmembrane (TM) as well as in soluble forms. Two membrane isoforms of the murine type II Fc gamma R, Fc gamma RIIb1 and Fc gamma RIIb2, have been described. They result from the translation of alternatively spliced pre-mRNA, Fc gamma RIIb2 lacking sequences of the first intracytoplasmic domain (IC1). Soluble forms of Fc gamma R (sFc gamma R) have previously been shown to result from proteolysis of membrane receptors. We report here the identification, in macrophages, of a mRNA derived from the Fc gamma RII gene by splicing exons encoding the TM and IC1 domains, i.e. corresponding to a TM-deleted Fc gamma RIIb2 mRNA. A soluble protein possibly encoded by this mRNA was identified in macrophage supernatants. In accordance with Fc gamma R nomenclature, we propose to name this new Fc gamma RII isoform Fc gamma RIIb3. It is the most abundant sFc gamma R present in serum, as compared with sFc gamma R resulting from cleavage of membrane Fc gamma R.

Soluble Fc gamma R (sFc gamma R): detection in biological fluids and production of a murine recombinant sFc gamma R biologically active in vitro and in vivo.

Soluble forms of receptors for the Fc portion of IgG (sFc gamma R) were detected in biological fluids from mice and humans. In mouse bearing tumors, circulating amounts of sFc gamma R increased concurrently with tumor growth. Tumors secreting IgG2a, IgG2b or IgG3 led to a 5- to 10-fold increase in serum sFc gamma R levels whereas tumors secreting IgG1, IgGA or other types of tumors (non Ig B cell tumors, T cell lymphoma and a melanoma) increased 2- to 3-fold the levels of circulating sFc gamma R. In the human, sFc gamma R were also detected in whole unstimulated saliva. Levels of sFc gamma RII and of sFc gamma RIII were variable and did not seem to depend on the dental status of the individuals. Finally, a murine recombinant sFc gamma R (rsFc gamma R) composed of the two extracellular domains of Fc gamma RII was produced by culture of transfected L cells in bioreactors. The purified rsFc gamma R was found to inhibit antibody production in vitro in anti-SRBC responses and by cultures of small B cells stimulated by anti-IgM antibodies in the presence of IL-4 and IL-5. Moreover, the i.p. injection of this material into adult mice immunized with SRBC led to a decrease of IgG antibody production by splenocytes, as measured by a hemolytic plaque assay, and in serum, as measured by antigen-specific ELISA.

Murine soluble Fc gamma receptors/IgG-binding factors (IgG-BF): analysis of the relation to Fc gamma RII and production of milligram quantities of biologically active recombinant IgG-BF.

The production of soluble forms of low-affinity Fc gamma R by cells expressing recombinant or natural membrane Fc gamma RII, and the structural relationships between these soluble receptors and membrane Fc gamma RII are described. We show that 37-40 kD soluble Fc gamma RII, corresponding to the two N-terminal domains of Fc gamma RII and binding to IgG, are spontaneously produced in vitro by cleavage of membrane Fc gamma RII. Moreover, we describe methods to produce and purify to homogeneity large quantities of endotoxin-free recombinant IgG-binding factor (rIgG-BF) from the culture medium of a cell line transfected with a mutated Fc gamma RII cDNA. These methods include the use of bioreactors for culturing transfected fibroblasts and the purification of rIgG-BF by ion-exchange chromatography and hydrophobic-interaction chromatography. By using such procedures, about 2.4 mg of rIgG-BF were purified from 1 liter of culture medium of transfected fibroblasts. Like natural IgG-BF, the 95-99% pure rIgG-BF suppressed, in a dose-dependent manner, secondary in vitro IgG antibody responses to sheep red blood cells.

Recombinant soluble receptors for the Fc gamma portion inhibit antibody production in vitro.

The problem of the structural relationship between suppressive IgG-binding factor and low-affinity receptors for the Fc portion of IgG (Fc gamma RII) has not yet been solved. In the present work we have isolated a recombinant soluble Fc gamma RII containing only the two external domains of Fc gamma RII, and analyzed its biochemical characteristics and biological activity. A cDNA encoding Fc gamma RII was mutated by the creation of a stop codon at the Lys175 codon. L cells have been transfected with this cDNA inserted into an expression vector. A cell line was obtained that secretes recombinant soluble Fc gamma RII which reacts with a monoclonal anti-Fc gamma RII antibody and binds to IgG1, IgG2a and IgG2b murine isotypes but not to IgG3 or F(ab')2 fragments of IgG2a. The secreted molecule contains two molecular species of relative molecular mass (Mr) 44,000 and 34,000-38,000 and of pI 4.5 and 6.3. They correspond to different glycosylations of a single polypeptide of Mr 19,000. After purification to homogeneity, soluble Fc gamma RII has found to suppress secondary and primary in vitro antibody responses in a dose-dependent way. The present work shows that recombinant soluble Fc gamma RII has biochemical characteristics, immunoreactivity and biological activity similar to those of suppressive IgG-binding factor.

Identification of the Fc gamma RII-related component of murine IgG-BF.

Suppressor murine IgG-BF produced by the T cell hybrid (T2D4) expressing low affinity Fc gamma R (Fc gamma RII) contain four biologically active polypeptides of pI 5.2, 6.3, 7.7 and 8.3, respectively. They were fractionated by affinity chromatography on immunoadsorbents coupled with F(ab')2 fragments of the monoclonal anti-Fc gamma RII antibody 2.4G2 and by hydrophobic interaction chromatography. Both methods led to the identification of biologically active IgG-BF which react with 2.4G2 and of IgG-BF which do not react with 2.4G2. Molecules bearing the epitope recognized by 2.4G2 had an apparent pI of 5.3 while the pI of those which did not express this epitope were 6.3, 7.8 and 8.5, respectively. Therefore, one IgG-BF polypeptide of pI 5.3 is probably related to Fc gamma RII.

The carbohydrate moieties of suppressor IgG-binding factor released by murine T cells.

The carbohydrate moieties of murine IgG-binding factor (IgG-BF) were studied using lectins binding N-glycosylated sequences such as Concanavalin A (Con A), Lens culinaris agglutinin (LcA), and wheat germ agglutinin (WGA), and lectins binding O-glycosylated sequences such as peanut agglutinin (PNA) and Helix pomatia Agglutinin (HpA). Sources of IgG-BF were: (1) supernatants from T2D4, a T cell hybridoma constitutively producing IgG-BF, and (2) factor purified by affinity chromatography on rabbit IgG-Sepharose, using T2D4 supernatants or supernatants of alloantigen-activated T cells (ATC) as starting material. The presence of IgG-BF was assessed by its ability to inhibit secondary anti-sheep red blood cell (SRBC) IgG antibody responses in vitro and to inhibit rosette formation between Fc gamma receptor (Fc gamma R)-positive spleen cells and erythrocytes sensitized with rabbit anti-Forssman IgG antibodies. Fractionation on immobilized lectins showed that IgG-BF: (1) is completely adsorbed by WGA and PNA and partially by Con A, LcA and HpA, and (2) can be eluted from the five different lectins using the competitor sugars. When produced in the presence of tunicamycin, an inhibitor of N-glycosylation, IgG-BF still binds to HpA which has affinity for O-glycosylated carbohydrate chains. These results indicate that IgG-BF is a glycoprotein with N- and O-glycosylated carbohydrate moieties.

Size and charge heterogeneity of murine IgG-binding factors (IgG-BF).

Size and charge of murine IgG-binding factors (IgG-BF) were determined. Four different sources were used to produce the factors: a) cells of a T cell hybrid (T2D4) constitutively secreting IgG-BF upon incubation in serum-free medium, b) T2D4 cells incubated with mouse monoclonal IgG1 antibody in order to induce in vitro the production of isotype-specific IgG1-BF, c) T2D4 cells induced in vivo by passage as ascites in nude mice and incubated in serum-free medium, and d) in vivo alloantigen-activated T cells (ATC) incubated in serum-free medium. IgG-BF were affinity purified on Sepharose beads coated with rabbit or mouse IgG and identified by their biologic activities, i.e., inhibition of in vitro secondary IgG antibody production to SRBC and inhibition of rosette formation between Fc gamma receptor-positive spleen cells and rabbit IgG-sensitized erythrocytes. IgG-BF produced by either of these cell sources was found to be heterogeneous in both size and charge. In each case, IgG-BF activities were recovered in three fractions of apparent Mr-74,000 to 78,000, 35,000 to 40,000, and 19,000 to 23,000-and in four fractions of pI-4.7 (or 5.3, depending on experimental conditions), 6.5, 7.7, and 8.4. Moreover, IgG-BF translated in vitro from T2D4 poly A RNA by using rabbit reticulocyte lysate exhibited the same heterogeneity. Thus, IgG-BF contain different proteins exerting similar biologic activities.

Intra-lesional injection of immunostimulants in bilateral rat tumors.

Rats grafted with either two or six fragments of isogenic methylcholanthrene-induced transplantable fibrosarcomas were treated IT1 with living BCG or killed C. parvum or a mixture of both immunostimulants. Various tumor combination and therapeutic agent dosages were compared. When two fragments of the same tumor McFiFi2 (S) were grafted simultaneously, IT treatment of one of these with 2 mg of BCG induced cure of both in 50% of the animals, but IT injection of 2 X 10(9) C. parvum was completely without effect in this situation. The prognosis was however, improved when the dose of immunostimulant was increased. The best results were obtained when each individual tumor in rats with two or six simultaneous identical grafts were treated with a mixture of BCG and C. parvum. The combination of IT immunostimulant treatment and surgical excision of the treated lesion demonstrated a persistence of the curative effect on the remote untreated tumor. Double grafting with isologous non-identical tumors revealed the influence of tumor burden and of the specificity of anti-tumor action of the treatment. The distant specific regression obtained in this system implies a specific immunological mechanism mediated by effector cells and/or antibodies which can circulate, identify the target cell and destroy it. This is in accordance with morphological observations. The intimate contact between BCG and the growing structured tumor is necessary for the therapeutic phenomenon.