sFc Gamma Research Articles

Determination of the stoichiometry of protein complexes using liquid chromatography with fluorescence and mass spectrometric detection of fluorescently labeled proteolytic peptides.

A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFc gamma RIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex.

Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.

Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress.

Binding of the soluble, truncated form of an Fc receptor (mouse Fc gamma RII) to membrane-bound IgG as measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy has been used to investigate the binding of the soluble extracellular domain of mouse Fc gamma RII (sFc gamma RII) to an anti-trinitrophenyl monoclonal mouse IgG2b (GK14.1) specifically bound to substrate-supported planar membranes composed of dipalmitoylphosphatidylcholine (DPPC) and trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-cap-DPPE). The equilibrium dissociation constants for sFc gamma RII at GK14.1-coated TNP-cap-DPPE/DPPC planar membranes containing 0.5-25 mol% TNP-cap-DPPE were approximately 1 microM. Total internal reflection with fluorescence photobleaching recovery was used to examine the dissociation kinetics. The fluorescence recovery curves were better described as a sum of two exponentials rather than by one exponential; the rates and fractional recoveries were approximately 1 s-1 (65%) and approximately 0.1 s-1 (35%). The similarity between the values of these equilibrium and kinetic parameters to those previously measured for the binding of IgG in solution to intact mouse Fc gamma RII reconstituted into planar membranes suggests that conformational changes which may occur when IgG is constrained to a membrane surface do not significantly affect the equilibrium or kinetics of IgG-mouse Fc gamma RII binding. The stoichiometry of sFc gamma RII-GK14.1 binding was 1:4, indicating that a significant fraction of the membrane-bound antibodies were not accessible for receptor binding. Possible mechanisms that might underlay the observed heterogeneity in sFc gamma RII-IgG binding kinetics are discussed.

Soluble CD16/Fc gamma RIII induces maturation of dendritic cells and production of several cytokines including IL-12.

Fc gamma RIII (CD16), a low affinity FcR which binds IgG-containing immune-complexes, exists under membrane-associated forms and under a soluble form (sFc gamma RIII). The latter, present in biological fluids (serum, saliva), is generated by proteolytic cleavage of the two membrane-associated Fc gamma RIII isoforms, Fc gamma RIII-A (expressed by macrophages and NK cells) and Fc gamma RIII-B (expressed exclusively by neutrophils). Herein we demonstrate that dendritic cells (DCs), generated by culturing monocytes with GM-CSF and IL-4, bind biotinylated recombinant sFc gamma RIII. This binding is specific and involves the complement receptor CR3 (CD11b/CD18) and CR4 (CD11c/CD18). Indeed, preincubation of DCs with anti-CD11b and anti-CD11c mAbs decreased by 52% and 62% respectively the binding with sFc gamma RIII. Moreover, electron microscopy showed that binding of gold-labeled sFc gamma RIII to DCs maintained at 4 degrees C occurred within clathrin-coated pits. Once internalized, at 37 degrees C, sFc gamma RIII entered the endocytic pathway and reached the MHC class II compartments. Furthermore, DCs incubated for 48 h with multivalent sFc gamma RIII expressed increased levels of CD40, CD80, CD86, CD54, CD58, HLA class I and class II molecules and decreased levels of CD23 and CD32. These effects result in an increased capacity of DCs to trigger proliferative responses by CD4+ CD45RA+ allogeneic T cells. RT-PCR amplification demonstrated that incubation of DCs for 20 h in the presence of multivalent sFc gamma RIII induced the appearance of GM-CSF and IL-12 p40 mRNA. Among the cytokines constitutively expressed, IL-1 beta and IL-8 were strongly up-regulated whereas IL-6 and IL-12 p35 mRNA were increased to a lesser extent and the expression of MIP-1 alpha mRNA remained constant. Finally, ELISA tests demonstrated that DCs incubated with multivalent sFc gamma RIII secreted the cytokines IL-1 beta, IL-6, IL-8, GM-CSF and IL-12 p75. Thus, while becoming internalized sFc gamma RIII could affect the capacity of DCs to present antigens and, via the induction of accessory molecules and the release of the IL-12 p75 protein, could initiate Th1 type immune response.

The use of rh‐G‐CSF in chronic autoimmune neutropenia: reversal of autoimmune phenomena, a case history

An 8-year-old boy had been suffering from chronic autoimmune neutropenia for more than 5 years. The neutropenia proved to be resistant to high-dose steroids and intravenous (either low-or high-dose) immunoglobulin (Ig) therapy. The chronic autoimmune thrombocytopenia and recurrent phases of autoimmune haemolytic anaemia did, however, respond to high-dose prednisone. Other signs of immune dysregulation in this patient consisted of insulin-dependent diabetes mellitus type I (IDDM) and an acquired hypogammaglobulinaemia, most compatible with common variable immunodeficiency (CVI). Prior to rhG-CSF therapy the child had suffered for more than 2 years from recurrent life-threatening bacterial infections. Anti-neutrophil autoantibodies had pan-Fc gamma RIII (CD116, NA1/NA2) specificity. The neutropenia as well as the antineutrophil autoantibodies disappeared when subcutaneous rhG-CSF therapy was started. Upon tapering rhG-CSF, anti-Fc gamma RIII antibodies reappeared together with an absolute neutropenia. Renewed administration resulted again in the normalization of symptoms. Soluble Fc gamma RIII (sFc gamma RIII) antigen levels in plasma increased dramatically during rhG-CSF treatment. These high levels of sFc gamma RIII together with increased numbers as well as decreased apoptotic reactions of neutrophils apparently result in adsorption of the autoantibodies in vivo, contributing to the normalization of autoimmune-mediated neutropenia upon rhG-CSF treatment. Long-term administration of rhG-CSF represents as alternative in the treatment of autoimmune neutropenia.

NA-phenotype-dependent differences in neutrophil Fc gamma RIIIb expression cause differences in plasma levels of soluble Fc gamma RIII.

Soluble Fc gamma RIII in plasma is primarily derived from neutrophils and is a measure of the total body neutrophil mass. We have developed a new, sensitive 'sandwich' ELISA to measure soluble Fc gamma RIII in plasma and released Fc gamma RIII in cell supernatants. Both sFc gamma RIIIa, derived from NK cells and sFc gamma RIIIb, derived from neutrophils are detected in the assay. However, plasma analysis of Fc gamma RIIIB gene-deficient donors suggested that sFc gamma RIIIa contributes only marginally to the total amount measured in healthy individuals. Furthermore, we observed that plasma of homozygous NA1-positive donors contained lower amounts of sFc gamma RIII than plasma of homozygous NA2-positive donors. Heterozygous donors were found to have intermediate levels of sFc gamma RIII in their plasma. Hemizygous Fc gamma RIIIB gene-deficient donors were found to have half the amount of sFc gamma RIII in their plasma compared to donors with two Fc gamma RIIIB alleles. These NA phenotype-dependent differences in plasma sFc gamma RIII could not be contributed to either an assay artefact or NA-dependent differences in shedding of Fc gamma RIIIb upon neutrophil activation. Calibration curves constructed with plasma of homozygous donors did nor reveal NA-dependent differences in antibody affinity. Measurement of released Fc gamma RIIIb in supernatants of neutrophils stimulated with PMA, and inhibition of this signal with human IgG revealed no NA-dependent differences. However, NA-dependent differences in neutrophil Fc gamma RIIIb expression were present, comparable to the differences found in plasma levels of sFc gamma RIII. Differences in the amounts of released Fc gamma RIII in supernatants of NA-typed apoptotic neutrophils were similar to initial differences in Fc gamma RIIIb expression, again being lower in NA1-positive than in heterozygous and NA2-positive donors. In conclusion, NA-dependent differences in plasma levels of soluble Fc gamma RIII seem to be caused by differences in expression of the receptor on the neutrophil membrane.

Soluble Fc gamma receptor III (CD 16) and eicosanoid concentrations in gut lavage fluid from patients with inflammatory bowel disease: reflection of mucosal inflammation.

Activated neutrophils cause tissue injury in inflammatory bowel disease (IBD). Upon activation, they shed soluble Fc gamma IIIb receptors (sFc gamma RIIIb). The subsequent inflammatory response is modulated by several mediators, including neutrophil derived leukotriene B4 (LTB4), thromboxane B2 (TXB2), and prostaglandin E2 (PGE2). The aim of this study was to determine the value of gut lavage sFc gamma RIII and eicosanoid measurements for the assessment of mucosal inflammation in IBD. A total of 18 patients with active IBD, 10 ulcerative colitis (UC), and eight Crohn's disease (CD), and 12 control patients underwent whole gut lavage. Disease activity, endoscopic appearance, and histopathology were graded. Samples were processed for the determination of sFc gamma RIIIb, LTB4, PGE2, and TXB2. Soluble Fc gamma RIIIb concentrations were increased in both IBD groups. Significant correlations were seen between sFc gamma RIIIb and LTB4 values with histology scores. Mean eicosanoid lavage fluid concentrations in control patients were 14.1 pg/ml for LTB4, 5.6 pg/ml for PGE2, and 397 pg/ml for TXB2. Concentrations of all eicosanoids in IBD patients were significantly increased: LTB4 in UC: mean 73.2 pg/ml, in CD: 96.4 pg/ml (both p < 0.01 v controls). PGE2 in UC: 20.2 pg/ml, in CD: 43.4 pg/ml (p < 0.01). TXB2 in UC: 719.3 pg/ml, in CD: 180.6 pg/ml (both p < 0.05). Whole gut lavage fluid analysis is an effective method to study mucosal eicosanoid production. Soluble Fc gamma RIIIb concentrations in gut lavage fluid closely correlate with histological signs of mucosal inflammation and with lavage LTB4 concentration. These data suggest that lavage Fc gamma RIIIb assessment may be used as a simple assay to estimate mucosal neutrophil infiltration in IBD.

Membrane and soluble Fc gamma RII/III modulate the antigen-presenting capacity of murine dendritic epidermal Langerhans cells for IgG-complexed antigens.

Murine dendritic epidermal Langerhans cells (LC) are APC. This implies that LC take up, process, and present Ag to T cells. One way of doing so that could allow Ag internalization is provided by the low affinity receptors for the Fc region of IgG (Fc gamma R), which murine LC are known to express, although their isoform(s) and function(s) have not been defined. By using molecular biology and biochemical approaches, we demonstrated that LC expressed Fc gamma RIIb2 and Fc gamma RIII. Furthermore, LC internalized Fc gamma R by receptor-mediated endocytosis, as observed with gold-labeled anti-Fc gamma RII/III mAb or immune complexes. We demonstrated the biologic relevance of this process by observing that Fc gamma R-mediated Ag internalization improved by approximately 300-fold the Ag-presenting capacity of LC to T cells. Moreover, analysis of cell culture supernatants showed that two forms of soluble Fc gamma R (sFc gamma R) were released by LC: the first most probably was the secreted transmembrane-deleted Fc gamma RII isoform, Fc gamma RIIb3, and the second was a soluble receptor probably derived from the membrane-associated Fc gamma RII/III. The ability of two recombinant forms, corresponding to the two sFc gamma R released by LC, to inhibit Fc gamma R-mediated presentation enhancement was assayed. Preincubation of IgG-complexed Ag with either rsFc gamma R led to a dose-dependent decrease in the Ag-presenting capacity of LC. Taken together, our results suggest that, in vivo, LC express membrane Fc gamma R, which increase their Ag-presenting capacity for IgG-complexed Ag, and release sFc gamma R, which might be able to modulate this Ag presentation.

The plasma concentration of soluble Fc-gamma RIII is related to production of neutrophils.

Fc gamma RIII (the CD16-antigen), a low-affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes and macrophages. A soluble form of Fc gamma RIII has been identified in human plasma. This soluble form of Fc gamma RIII (sFc gamma RIII) originates from release by neutrophils. In the present study we show by transfusions of plasma that contains sFc gamma RIII of one allotype (NA1-Fc gamma RIII) in recipients homozygous for the other allotype (NA2-Fc gamma RIII) that the clearance of sFc gamma RIII is about 0.7 ml/min. Because the concentration of sFc gamma RIII was found to be constant in a small cohort of donors followed for about 1.5 years, the half-life of NA1-sFc gamma RIII is about 1.8 d, assuming a one-compartment model. The plasma concentration of sFc gamma RIII depended mainly on the production of neutrophils in the bone marrow, and was not influenced by shifts of neutrophils from one pool to another (storage, marginating or circulating pool). Because Fc gamma RIII is only expressed on mature neutrophils, this implies that the concentration of sFc gamma RIII depends on production of mature neutrophils.

Soluble Fc gamma RIIIa is present in plasma and is derived from natural killer cells.

Fc gamma RIII (CD16), a receptor for complexed IgG, is encoded by two very homologous genes: Fc gamma RIIIA and Fc gamma RIIIB. NK cells and macrophages express Fc gamma RIIIa, whereas only neutrophils constitutively express Fc gamma RIIIb. In a previous study we found that soluble (s)Fc gamma RIII in plasma seemed to originate only from neutrophils. However, CD16 mAb, directed against different epitopes of Fc gamma RIII, precipitated a glycoprotein from plasma of homozygous Fc gamma RIIIB gene-deficient donors. This glycoprotein migrated in a similar way as did released Fc gamma RIIIa derived from NK cells, whereas Fc gamma RIIIa released by cultured monocytes migrated differently and appeared to be more heavily glycosylated on SDS-PAGE. After deglycosylation, the M(r) of the plasma sFc gamma RIIIa was similar to that of released Fc gamma RIIIa. Moreover, V8-protease maps were identical. Therefore, we conclude that sFc gamma RIIIa is also present in plasma and is derived from NK cells. Because sFc gamma RIII levels are hardly detectable in the plasma of most homozygous Fc gamma RIIIB gene-deficient donors, we suspect that the sFc gamma RIIIa level is negligible compared with the level of sFc gamma RIIIb in plasma of healthy donors. Two patients with an NK cell lymphocytosis had a high plasma level of sFc gamma RIIIaNK. Furthermore, high levels of sFc gamma RIIIaNK were found in plasma of two patients with rheumatoid arthritis. Thus, the level of sFc gamma RIIIaNK might reflect either an increase in circulating NK cells or an enhanced release of Fc gamma RIIIaNK in certain diseases. This study shows that an assay that discriminates between sFc gamma RIIIa and sFc gamma RIIIb is necessary for the interpretation of sFc gamma RIII levels in patients.

Human epidermal Langerhans cells secrete a soluble receptor for IgG (Fc gamma RII/CD32) that inhibits the binding of immune complexes to Fc gamma R+ cells.

Langerhans cells (LC) express Fc gamma RII on their cell surface. In this paper, we demonstrate that these cells also release soluble Fc gamma RII (sFc gamma RII) molecules. LC express transcripts encoding a membrane-associated receptor and a transmembrane-deleted Fc gamma RIIA. The latter form was identified in LC culture supernatants using specific antibodies. CHO cells, transfected with LC-derived cDNA encoding the transmembrane-deleted Fc gamma RIIA, secrete sFc gamma RIIA that include the intracellular domain and exhibit the same backbone as the protein identified in LC supernatants. Secreted sFc gamma RIIA exhibits the same pattern of binding to human and mouse IgG subclasses as do membrane Fc gamma RII and inhibits the binding of immune complexes to Fc gamma RII+ cells. In addition, CHO cells expressing the membrane-associated Fc gamma RIIA release truncated and unstable Fc gamma RIIA molecules that lack the intracellular domain. Thus, sFc gamma RII can result from shedding of membrane molecules and/or from secretion of soluble receptors lacking the transmembrane domain.

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.