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 Fcgamma receptor type III (FcgammaRIII, CD16) triggers cell activation through interaction with complement receptors.

The type III-B Fcgamma receptor (FcgammaRIII-B) is a glycosyl-phosphatidylinositol-linked receptor found on human neutrophils. A soluble form of FcgammaRIII-B (sCD16) corresponding to the extracellular region of the receptor circulates in plasma. In the present work, we have identified membrane receptors for sCD16. Soluble CD16 bound to CR3 (CDllb/CD18)- and CR4 (CDllc/CD18)- positive leukocytes and cell lines, the labeling was inhibited by anti-CD11b, CD11c or CD18 mAbs, and the up-regulation of CR3 and CR4 led to an increased fixation of sCD16. Transfected eukaryotic cells expressing recombinant CD11b/CD18 or CD11c/CD18 heterodimers but not those expressing CD11a/CD18 bound sCD16. Moreover, the lectin-like binding site of CR3 is probably involved in the interaction with sCD16, as suggested by inhibition studies using mAbs against CR3 or sugars such as N-acetyl D-glucosamine, alpha- or beta-methyl D-glucoside, alpha- or beta-methyl D-mannoside, or zymosan. Thus, the complement receptors CR3 and CR4 are membrane receptors for sCD16. Through this interaction, sCD16 induces a CR3-dependent production of IL-6 and IL-8 by monocytes. These results suggest that sCD16 plays a regulatory role in inflammatory processes and provide a molecular basis for the interaction between FcgammaRIII-B and CR3 described on the cell membrane.

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.

Soluble CD16 binds peripheral blood mononuclear cells and inhibits pokeweed-mitogen-induced responses.

Human neutrophils express two types of low affinity receptors for IgG, Fc gamma RII or CD32 and Fc gamma RIIIB or CD16. Human serum contains soluble CD16 (sCD16), which is produced by proteolysis of neutrophil Fc gamma RIIIB, the cleavage site being located close to the cell surface. In order to assess the functional roles of sCD16, we have produced, in eukaryotic cells, a recombinant sCD16 containing the extracellular region of Fc gamma RIIIB. Purified sCD16, of molecular mass of 48 kD, bound human IgG1 and IgG3 but not IgG2, IgG4, or F(ab')2. It inhibited, in a time and dose-dependent fashion, proliferation and IgM and IgG production of human peripheral blood mononuclear cells (PBMC) stimulated by pokeweed mitogen (PWM) in vitro. FACS analysis showed that biotinylated sCD16 bound specifically to a fraction (35%) of PBMC, which corresponds to monocytes and to subsets of B and T lymphocytes. Moreover, sCD16 did not modify the staining of PBMC by FITC-coupled PWM. Thus, the biologic function(s) of sCD16 on PWM-induced responses are exerted through direct and specific interaction(s) with mononuclear blood cells and not with PWM. In conclusion, neutrophils may play a regulatory role on immune responses via the production of soluble forms of CD16 with cell-binding and antiproliferative capacities.

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.

6"'-Deamino-6"'-hydroxy derivatives, as intermediates in the biosynthesis of neomycin and paromomycin.

From broths of a neomycin producing Streptomyces fradiae and of a mutant of Streptomyces rimosus forma paromomycinus respectively, 6"'-deamino-6"'-hydroxyneomycin and 6"'-deamino-6"'hydroxyparomomycin were obtained and their structures established by mass and 13C-NMR spectroscopy and by the study of hydrolytic fragments. These new compounds, which are both present as two epimers at C-5"', are suggested as intermediates in the biosynthesis of the parent antibiotics. The place and the mechanism of the 5"'-epimerisation and of the 6"'-amination are discussed.

Interspecific recombination among aminoglycoside producing streptomycetes.

From auxotrophic double mutants of Streptomyces rimosus forma paromomycinus and Streptomyces kanamycerticus producing little or no antibiotic, stable prototrophic recombinants were obtained with low frequencies. Most of the recombinants differed from the parents in morphology and antibiotic production. The most frequent classes of recombinants behaved as streptomycetes of the "red" series and produced a wide range of neomycin yields, in contrast to the parents which produced paromomycin and a small proportion of neomycin, or kanamycin, respectively. Hypotheses on the nature of the genetic material exchanged are discussed.