Catherine Sautès

Soluble Fc gamma receptors.

Soluble FCγ receptors have been identified in biological fluids of mice and humans. They are produced either by alternative splicing of the exon encoding the transmembrane region of the receptor (FcγRII) or by proteolytic cleavage at the cell membrane (FCγRII and FC7RIII). They inhibit B cell proliferation and immunoglobulin production. Their concentrations in plasma seem to be modified during the development of certain diseases, as for instance in multiple myeloma, where plasma concentrations oi soluble FCγKIll are correlated with the stage of the disease.

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

CD16 (Fcγ R type III) is a low‐affinity IgG Fc receptor (R) that exists in two isoforms, a transmembrane FcγRIIIa expressed by NK cells and monocytes, and a phosphatidylinositol‐linked FcγRIIIb 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 FcγRIIIb 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.

The murine Fc-gamma (Fcγ) receptor type II B1 is a tumorigenicity-enhancing factor in polyoma-virus-transformed 3T3 cells

The murine receptor for the Fc portion of IgG is a molecule expressed by cells of the immune system. This study suggests the hypothesis that Fcγ receptor type II B1 (FcγRIIB1) functions as a progression‐enhancing factor when expressed ectopically on non‐lymphoid tumor cells. It has been shown previously that BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) do not express FcγRII but acquire the expression of this receptor following an in vivo passage in syngeneic mice. The specific FcγRII transcript present in tumor cells was identified in this report as FcγRIIB1 (B1). In order to determine whether or not the ectopically expressed FcγRII plays a role in the progression of these transformed cells, PyV‐transformed 3T3 cells were transfected with B1‐cDNA. The B1 transfected cells were tested for their ability to form local tumors in syngeneic mice, as compared to transfected cells which express the co‐transfecting neomycine resistance (neores) DNA alone or together with the lacZ gene. FcγRIIB1 expressors exhibited a significantly higher tumorigenic phenotype than FcR‐negative controls, though both types of cells exhibited the same growth curve in vitro. The ability of FcγRIIB1 to act as a potentially tumorigenicity‐enhancing factor was also demonstrated as FcγRII was expressed by tumor cells, originating from inoculated FcγRIIB1‐transfected cells, or from inoculation of a mixture of receptor‐positive and ‐negative cells. B1‐expressing cells dominated the tumor‐cell population over non‐expressors. This dominance strengthened the hypothesis that FcR plays a role in tumor progression in vivo.

Ligands and biological activities of soluble Fcγ receptors

Soluble forms of low-affinity Fc gamma receptors (sFc gamma R) circulate in biologic fluids. Their plasmatic levels vary in immunological disorders or related diseases. They are produced by enzymatic cleavage of the membrane receptors or by alternative splicing. They bind IgG with the same isotype specificity as their cell surface counterparts and thus modulate Fc-dependent immune functions. Recent data suggest that they also bind non-Ig ligands present on leukocytes. Functional implications of these findings are discussed.

Soluble CD16/FcγRIII 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.

Contribution of the intracellular domain of murine FC‐gamma receptor type IIB1 to its tumor‐enhancing potential

We have previously shown that Fc gamma receptor type II B1 (FcγRIIB1), when expressed on non‐lymphoid tumor cells, significantly enhanced their tumorigenic phenotype. This study elucidates the role of the intracellular domain of FcγRIIB1 in the enhancement of the malignant phenotype of polyoma‐transformed 3T3 cells. We investigated the tumorigenic potential conferred by different variants of the receptor: FcγRIIB1, a full‐length receptor (B1) whose intracellular region is encoded by exons 8, 9 and 10; FcγRIIB2, a spliced variant (B2) whose cytoplasmic domain comprises exons 9 and 10 and lacks exon 8; and FcγRIIB1‐CT53, a deleted mutant whose cytoplasmic domain contains the fragment encoded by exon 8 alone. We have investigated various properties of cells transfected with each of the above variants: tumorigenicity in syngeneic mice, formation of colonies in soft agar, growth rate, production of soluble receptor and capping of the ligand‐bound receptor. Results show that while the presence of exon 8 did not enhance growth rate in vitro or production of soluble FcγR, it did enhance the tumorigenic phenotype of transfected cells (both in vivo and in vitro growth in soft agar). B1‐expressing cells exhibited a significantly higher tumorigenic phenotype than B2 cells. The presence of exon 8 alone (CT53 mutant) conferred the transfected cells a higher tumorigenic phenotype than FcγR‐negative control cells but lower than intact B1 or B2 cells, indicating that the presence of B1‐specific exon 8 is not sufficient but that the presence of an intact B1 intracellular domain is essential, for conferring the high tumorigenicity phenotype upon cells. We conclude that the capping, following ligand binding contributed by exon 8, and the function contributed by the specific localization of exons 9 and 10 in B1 cells may determine their malignant phenotype.

Immunoglobulin G-binding factors (IgG-BF) inhibit IgG secretion by, as well as proliferation of, hybridoma B cells

Mouse immunoglobulin G-binding factors (IgG-BF) produced either by activated T cells (ATC) or by hybridoma T cells (T2D4) directly inhibit the in vitro IgG secretion by hybridoma B cells. This inhibition affects IgG1, IgG2a and IgG2b and can be detected as early as after 2 h incubation of the cells with IgG-BF eluted from non-equilibrium pH gradient electrophoresis gels. Moreover, IgG-BF also exert a strong growth-inhibitory effect on hybridoma B cells without any detectable immediate cytotoxicity. These results provide an experimental basis to analyze the molecular and biological effects induced by IgG-BF on B cells.

Mechanism of inhibition of lipopolysaccharide-stimulated mouse B-cell responses by transforming growth factor-β1

Transforming growth factor-beta 1 (TGF beta 1) is a pleiotropic cytokine which inhibits growth of many cell types and positively or negatively regulates the production of Ig isotypes. By using mouse resting B cells stimulated by lipopolysaccharide (LPS), we investigated whether the effect of TGF beta 1 on Ig production is related to its effect on cell growth. We show that low doses of TGF beta 1 stimulate IgG3 and IgG2b production whereas higher doses inhibit IgM, IgG3, IgG1 and IgG2b secretion and cell proliferation. TGF beta 1 titration curves and kinetics experiments suggested that the inhibitory effect on Ig secretion and B-cell growth are closely related. We defined the phase at which TGF beta 1 exerts its anti-proliferative effect on mouse B cells. TGF beta 1 does not modify the increase in expression of class II antigens which occurs before transition from G0 to G1. However, it partially inhibits the induction of expression of low-affinity Fc gamma RII and cell enlargement which both begin during the early G1 phase, and it totally blocks induction of the expression of transferrin receptors, a marker of the late G1 phase. Thus, TGF beta 1 blocks LPS-stimulated mouse B cells in the early G1 phase, and this results in inhibition of Ig production.

Soluble FcγR (sFcγR) : detection in biological fluids and production of a murine recombinant sFcγR biologically active in vitro and in vivo

Abstract Soluble forms of receptors for the Fc portion of IgG (sFcγR) were detected in biological fluids from mice and humans. In mouse bearing tumors, circulating amounts of sFcγR increased concurrently with tumor growth. Tumors secreting IgG2a, IgG2b or IgG3 led to a 5- to 10-fold increase in serum sFcγR 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 sFcγR. In the human, sFc7#x03B3;R were also detected in whole unstimulated saliva. Levels of sFcγRII and of sFcγRIII were variable and did not seem to depend on the dental status of the individuals. Finally, a murine recombinant sFcγR (rsFcγR) composed of the two extracellular domains of Fcγ RII was produced by culture of transfected L cells in bioreactors. The purified rsFcγR 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.

Immunoglobulin-binding factors

It has been known for more than a decade that lymphocytes [1] and also granulocytes [2] and macrophages [3] release molecules capable of binding immunoglobulin (Ig) isotypes. First IgG-binding factors (IgG-BF) [1], then IgE-BF [4], IgA-BF [5] and more recently IgD-BF [6], were reported. Soon after the discovery of IBF, three of their major properties were characterized: (i) they are produced by cells, mostly T lymphocytes, bearing Fc receptors (FcR) for the same Ig isotype; thus, IgG-BF are produced by Fcq/R positive cells, lgE-BF, IgA-BF and IgD-BF are released by FceR, FcaR and Fc6Rpositive cells, respectively; (ii) their production increases upon interaction of Ig with FcR, IgG inducing IgG-BF release [7], IgE and IgA inducing the production of IgE-BF and IgA-BF, respectively [5, 8]; when lymphocytes are incubated in serum-free buffer, the appearance of IBF in the supernatant parallels the shedding of membrane FcR [9]; (iii) IBF have been found to regulate Ig production in vitro, in a strictly isotype-specific manner for IgE-BF and IgA-BF, and in a broader way for IgG-BF (which regulates IgG and lgM responses). Moreover, some IBF have been reported to exert dual effects: IgE-BF produced by T cells either potentiated or suppressed IgE production, depending on their glycosylation [10]. Both suppressive and potentiating IgA-BF [5, 11] and suppressive IgG-BF [7, 12] have also been described. Taken together, these obervations led, soon after the description of IBF, to the proposal that these molecules were in fact shed FcR [13] (the term solu-