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.

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 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.