On the interaction between agalactosyl IgG and Fcγ receptors

One of the serum abnormalities observed in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is the occurrence of IgG that lacks the terminal galactose on asparagine-linked biantennary complex type oligosaccharides [Gal(0)-IgG] located in the CH2 domain. Additionally, IgG without glycosylation is known to be defective in several effector functions due to a reduced ability to bind to its specific receptors (FcγR). It has thus been speculated that, by analogy with unglycosylated IgG, Gal(0)-IgG may also be functionally impaired or exert altered effector mechanisms. If this were true, Gal(0)-IgG could contribute to the phenotype of above-mentioned autoimmune diseases, like impaired immune complex clearance and defective down-regulation of activated B cells. Here, we show by three different methods that the interaction of Gal(0)-IgG and normally glycosylated IgG with the low-affinity FcγRII (CD32) is indistinguishable with respect both to binding and receptor-mediated signalling. These data argue against a prominent role for FcγR-dependent Gal(0)-IgG interactions in the etiology or pathogenesis of autoimmune diseases.     

FcγRIIB regulation of BCR/TLR‐dependent autoreactive B‐cell responses

Crosslinking of Fc γ receptor II B (FcγRIIB) and the BCR by immune complexes (IC) can downregulate antigen‐specific B‐cell responses. Accordingly, FcγRIIB deficiencies have been associated with B‐cell hyperactivity in patients with systemic lupus erythematosus and mouse models of lupus. However, we have previously shown that murine IgG2a‐autoreactive AM14 B cells respond robustly to chromatin‐associated IC through a mechanism dependent on both the BCR and the endosomal TLR9, despite FcγRIIB coexpression. To further evaluate the potential contribution of FcγRIIB to the regulation of autoreactive B cells, we have now compared the IC‐triggered responses of FcγRIIB‐deficient and FcγRIIB‐sufficient AM14 B cells. We find that FcγRIIB‐deficient cells respond significantly better than FcγRIIB‐sufficient cells when stimulated with DNA IC that incorporate low‐affinity TLR9 ligand (CG‐poor dsDNA fragments). AM14 B cells also respond to RNA‐associated IC through BCR/TLR7 coengagement, but such BCR/TLR7‐dependent responses are normally highly dependent on IFN‐α costimulation. However, we now show that AM14 FcγRIIB^?/? B cells are very effectively activated by RNA IC without supplemental IFN‐α priming. These results demonstrate that FcγRIIB can effectively modulate both BCR/TLR9 and BCR/TLR7 endosomal‐dependent activation of autoreactive B cells.     

T lymphocyte development in the absence of Fcϵ receptor Iγ subunit: analysis of thymic-dependent and independent αβ and γδ pathways

During fetal development, early thymocyte progenitors transiently express low affinity Fc receptors for IgG (FcγR) of both FcγRII and III isoforms. Only the FcγRIII isoform requires association of an FcγRIII (CD16) α subunit with an FcϵRIγ homodimer for surface expression. To address the role of FcγR in ontogeny, we studied thymic development in FcϵRIγ^−/− mice. We find that day 14.5 CD4⁻CD8⁻ double-negative (DN) fetal thymocytes of FcϵRIγ^−/− mice express mRNA of both FcγRIIb₁ and FcγRIII. Surface expression of FcγRII/III is readily detected on these cells. It appears that FcγRIIb₁, whose surface expression is FcϵRIγ independent, replaces FcγRIII during thymic development in these animals. Moreover, subsequent development into CD4⁺CD8⁺ double-positive and CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive subsets appears normal even in the absence of FcϵRIγ. However, alterations were noted in adult animals among the DN αβ TCR⁺ thymocytes and peripheral splenic DN T cells as well as CD8αα⁺ intestinal intraepithelial lymphocytes (iIEL). In contrast to conventional T lymphocytes, which do not express either FcγRIII or FcϵRIγ, DN αβ TCR⁺ thymocytes and extrathymically derived αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL express TCR which incorporate FcϵRIγ as one of their subunits. Consistent with this, the TCR levels of these cells are lower than the TCR levels on cells from wild-type C57BL/6 mice. Despite the reduction in the level of surface TCR, the development of these cells was unaltered by the absence of FcϵRIγ. Thus, we observed alterations in adult DN αβ TCR⁺ thymocytes, splenic DN αβ TCR⁺ and DN γδ TCR⁺ large granular lymphocytes (LGL), and αβ TCR⁺ and γδ TCR⁺ CD8αα⁺β⁻ iIEL, but no detectable changes in their major fetal thymic developmental pathways. Cultivation of peripheral DN αβ TCR⁺ and DN γδ TCR⁺ cells from FcϵRIγ^−/− mice with interleukin-2 generates LGL which mediate natural killer activity. Unlike LGL from wild-type C57BL/6 mice, LGL from FcϵRIγ^−/− mice lack FcγRIII expression and could not mediate antibody-dependent cellular cytotoxicity through FcγRIII.     

FcγRIIa polymorphism in systemic lupus erythematosus (SLE): no association with disease

An allotypic variant of FcγRIIa, FcγRIIa-HR (FcγRIIa-R131), has been shown in vitroto reduce the capacity of phagocytic cells to bind and internalize IgG-containing immune complexes. Our aim was to determine whether this allotypic variant was associated with susceptibility to SLE and the development of lupus nephritis, as previous studies have suggested. FcγRIIA genotype analysis was performed by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 215 Caucasoid, 70 Afro-Caribbean, and 46 Chinese patients with SLE, and in 259, 77, and 49 ethnically matched controls, respectively. Distribution of FcγRIIa genotypes between the patients and ethnically matched controls was not significantly different in the three populations studied. No association between the FcγRIIa-HR allotype and nephritis was found. Our results suggest that the FcγRIIa-HR allotype is not a major factor predisposing to the development of SLE, or to lupus nephritis.     

Both FcγRIV and FcγRIII are essential receptors mediating type II and type III autoimmune responses via FcRγ‐LAT‐dependent generation of C5a

FcγRIV is a relatively new IgG Fc receptor (FcγR) that is reported to contribute to the pathogenesis of autoimmune diseases, although its specific role in relation to FcγRIII, complement and IgG2 subclasses remains uncertain. Here we define FcγRIV on macrophages as a receptor for soluble IgG2a/b complexes but not for cellular bound IgG2a and show that simultaneous activation of FcγRIV and FcγRIII is critical to mediate certain type II/III autoimmune responses. FcγRIII‐deficient mice display compensatory enhanced FcγRIV expression, are protected from lung inflammation after deposition of IgG complexes, and show reduced sensitivity to IgG2a/b‐mediated hemolytic anemia, indicating that increased FcγRIV alone is not sufficient to trigger these diseases in the absence of FcγRIII. Importantly, however, blockade of FcγRIV is also effective in inhibiting phagocytosis and cytokine production in IgG2b‐induced anemia and acute lung injury, processes that display a further dependence on C5a anaphylatoxin receptor. Using gene deletion and functional inhibition studies, we found that FcγRIII and FcγRIV are each essential to trigger an FcRγ‐linker for activation of T‐cell‐dependent signal that drives C5a production in the Arthus reaction. Together, the results demonstrate a combined requirement for FcγRIII and FcγRIV in autoimmune injury, and identify the linker for activation of T cells adaptor as an integral component of linked FcγR and C5a anaphylatoxin receptor activation to generate inflammation.     

FcγRI (CD64): An identity card for intestinal macrophages

Macrophages are becoming increasingly recognized as key cellular players in intestinal immune homeostasis. However, differentiating between macrophages and dendritic cells (DCs) is often difficult, and finding a specific phenotypic signature for intestinal macrophage identification has remained elusive. In this issue of the European Journal of Immunology, Tamoutounour et al. [Eur. J. Immunol. 2012. 42: 3150‐3166] identify CD64 as a specific macrophage marker that can be used to discriminate DCs from macrophages in the murine small and large intestine, under both steady‐state and inflammatory conditions. The authors also propose a sequential ‘monocyte‐waterfall’ model for intestinal macrophage differentiation, with implications for immune tolerance and inflammation at the gut mucosal interface. This Commentary will discuss the advantages and potential limitations of CD64 as a marker for intestinal macrophages.     

IgG-mediated anaphylaxis via Fcγ receptor in CD40-deficient mice

Anaphylaxis denotes an immediate hypersensitivity reaction to allergen, exclusively mediated by IgE antibodies. However, IgE antibodies do not explain all the syndromes that are encountered. We investigated potent IgG-mediated anaphylaxis in CD40-deficient mice that lack the immunoglobulin class switching for T cell-dependent antigens. Immunization with ovalbumin did not induce either humoral responses of IgG, IgA, and IgE, or systemic anaphylaxis in CD40-deficient mice. Although systemic anaphylaxis by active immunization was not observed in CD40-deficient mice, both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis assessed by mouse blood pressure monitoring with cervical artery catheterization did take place when antigen-specific IgG was transferred and then antigen challenge given. Further, to investigate the inflammatory pathway of IgG-mediated immediate hypersensitivity reactions, we focused on the Fcγ receptor (FcγR) function. Pretreatment of the mice with the anti-FcγRII/FcγRIII MoAb clearly blocked the response of PCA and passive systemic anaphylaxis, suggesting that they were initiated through FcγR. In conclusion, we directly demonstrate the IgG-mediated anaphylaxis and its triggering mechanism through FcγR in in vivo conditions. In addition to IgE-mediated anaphylaxis, IgG-mediated anaphylaxis should be considered and the blocking of FcγR would provide one of the therapeutic targets for the control of IgG-mediated hypersensitivity diseases.     

The β2 integrin Mac-1 but not p150,95 associates with FcγRIIA

In this study we have compared the ligand binding activity of the two closely related β2 integrins, Mac-1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac-1 has previously been shown to associate with FcγR, particularly FcγRIII, but K562 cells express only endogenous FcγRIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between FcγRIIA with Mac-1 and p150,95 in the absence of other FcγR. The main finding is that anti-FcγRII mAb have a profound inhibitory effect on cell adhesion mediated by Mac-1, but not on the adhesion mediated by p150,95. Thus, in spite of the fact that Mac-1 and p150,95 bind to the same or at least a very similar selection of ligands, their association with other receptors on the cellular membrane, and therefore their mode of regulation may be different.     

Co-localization of the neonatal Fcγ receptor and IgG in human placental term syncytiotrophoblasts

Transfer of maternal IgG through the human placenta furnishes the newborn with passive immunity to a number of infectious agents. The exact mechanism of this transfer is still unknown, but it is agreed that it involves active receptor-mediated transport. The neonatal Fc receptor is a major histocompatibility complex class I-like receptor originally identified in the intestines of newborn rodents. A similar receptor has recently been detected in human placental syncytiotrophoblasts. Using multilabeling fluorescence immunohistochemistry and confocal laser scanning microscopy, we found that the neonatal Fc receptor co-localizes with IgG and β2-microglobulin in granules of human placental syncytiotrophoblast. The Fc receptor is not detected on syncytiotrophoblast apical plasma membrane. Localization to the outermost cellular barrier between the fetal and maternal blood further strengthens the role of the Fc receptor in transplacental transport of IgG.     

The internalization of the IgG2a antigen receptor does not require the association with Ig-α and Ig-β but the activation of protein tyrosine kinases does

The B cell antigen receptor of class IgM is a multimeric protein complex containing the membrane-bound immunoglobulin molecule and a heterodimer of the two B cell-specific transmembrane proteins Ig-α and Ig-β. The B cell antigen receptor fulfills a dual role on the surface of B cells. First, it is a signal transduction complex which can activate protein tyrosine kinases and induce the release of Ca²⁺ ions from intracellular stores. Second, its internalization mediates the specific uptake of bound antigens, which are processed intracellularly and presented as major histocompatibility complex-bound peptides on the cell surface. In case of the IgM antigen receptor, the association with the heterodimer is necessary for expression of large amounts of IgM on the surface. We show here that the IgG2a antigen receptor can be expressed on the surface of myeloma cells in two structurally different forms: either with or without the Ig-α/Ig-β heterodimer. A functional comparison of the two forms of antigen receptors demonstrates that the Ig-α and Ig-β molecules are required for the activation of protein tyrosine kinases after cross-linking of the B cell antigen receptor. In contrast, both forms of IgG2a are equally well internalized. This suggests that Ig-α and Ig-β are essential for signal transduction through the IgG2a antigen receptor, whereas internalization can occur independently of the heterodimer.     

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum

During phagocytosis, surface receptors on neutrophils interact with pathogens opsonized with complement factor C3b/iC3b and in some cases with antibodies. In human immune sera antibodies directed against surface-bound M proteins mediated killing of Streptococcus pyogenes by neutrophils. Surprisingly, blocking of the Fc receptors had little effect on the killing. In contrast, inhibition of C3b/iC3b generation, or blocking of the major neutrophil iC3b receptor CD11b/CD18, enabled S. pyogenes to grow efficiently in immune sera. Inhibition of CD11b/CD18, but not of CD32, the major neutrophil signaling Fc receptor, prevented Streptococcus-induced NADPH oxidase-dependent respiratory burst, and blocking of C3b/iC3b formation inhibited Streptococcus-induced activation of Cdc42, a small GTPase critically involved in transmitting pro-inflammatory signals to the cytoskeleton. Consequently, ligation of CD11b/CD18 by bacteria-bound iC3b is necessary for inducing a neutrophil response leading to elimination of S. pyogenes in immune human serum.     

Racially restricted contribution of immunoglobulin Fcγ and Fcγ receptor genotypes to humoral immunity to human epidermal growth factor receptor 2 in breast cancer

Tumour‐associated antigen human epidermal growth factor receptor 2 (HER2) is over‐expressed in 25–30% of breast cancer patients and is associated with poor prognosis. Naturally occurring anti‐HER2 antibody responses have been described in patients with HER2 over‐expressing tumours. There is significant interindividual variability in antibody responsiveness, but the host genetic factors responsible for this variability are poorly understood. The aim of the present investigation was to determine whether immunoglobulin genetic markers [GM (genetic determinants of γ chains)] and Fcγ receptor (FcγR) alleles contribute to the magnitude of natural antibody responsiveness to HER2 in patients with breast cancer. A total of 855 breast cancer patients from Japan and Brazil were genotyped for several GM and FcγR alleles. They were also characterized for immunoglobulin (Ig)G antibodies to HER2. In white subjects (n = 263), GM 23‐carriers had higher levels of anti‐HER2 antibodies than non‐carriers of this allele (p = 0·004). At the GM 5/21 locus, the homozygotes for the GM 5 allele had higher levels of anti‐HER2 antibodies than the other two genotypes (P = 0·0067). In black subjects (n = 42), FcγRIIa‐histidine/histidine homozygotes and FcγRIIIa‐phenylalanine/valine heterozygotes were associated with high antibody responses (P = 0·0071 and 0·0275, respectively). FcγR genotypes in white subjects and GM genotypes in black subjects were not associated with anti‐HER2 antibody responses. No significant associations were found in other study groups. These racially restricted contributions of GM and FcγR genotypes to humoral immunity to HER2 have potential implications for immunotherapy of breast cancer.     

Expression of FcγRIII defines distinct subpopulations of fetal liver B cell and myeloid precursors

We have isolated four distinct fetal liver (FL) populations based on the expression of AA4.1 and the low-affinity Fcγ receptors type II and III (FcγRII/III), and characterized them with respect to B cell, T cell, and myeloid precursor content. Polymerase chain reaction analysis revealed that the prevalent FcγR isoform at this stage of FL development (day 12 of gestation) was FcγRIII. Two of the four populations, one which expressed AA4.1 but little if any FcγRII/III (AA4.1⁺), and one which expressed abundant levels of both markers (AA4.1⁺/ FcR⁺), contained B cell precursors that grew and differentiated to generate V_HDJ_H-rearranged B-lineage cells on S-17 stromal cells in the presence of IL-7. When cultured on FLST2 stromal cells only the AA4.1⁺ cells generated V_HDJ_H-rearranged B-lineage cells. T cell precursors as assayed by their ability to repopulate fetal thymi in organ culture were found only in the AA4.1⁺ fraction. In contrast to the lymphoid precursors, myeloid precursors able to generate colonies in methyl cellulose cultures were found in all four fractions including the one which expressed FcγRII/III but no AA4.1 (FcR⁺) and the one which expressed neither marker (AA4.1^?/FcR^?). The AA4.1⁺ population which contained both B cell and T cell precursors was enriched for precursors from many myeloid lineages including the most immature ones which generated multilineage colonies. In contrast, the AA4.1⁺/FcR⁺ population, which also contained B cell precursors, was almost devoid of myeloid precursors and the few that were detected were committed to the macrophage lineage. The population defined as FcR⁺ was also enriched for precursors; however, the majority of these were committed to the erythroid, the macrophage and the mast cell lineage. The fourth population which expressed neither marker (AA4.1^?/FcR^?) was enriched for relatively mature erythroid precursors which were not present in any of the other fractions. Together, these findings demonstrate that fractionation of FL cells on the basis of AA4.1 and FcγRII/III expression distinguishes subpopulations of B cell and myeloid precursors and suggests that the low-affinity FcγRIII could play a role in the development of early hematopoietic cells at this stage of ontogeny.     

Modulation of Fcγ receptor-mediated early events by the tyrosine phosphatase CD45 in primary human monocytes. Consequences for interleukin-6 production

Stimulation of primary human monocytes from several donors by cross-linking of Fcγ receptor type I (FcγRI) and FcγRII gave rise to calcium mobilization and protein tyrosine phosphorylation. These early events were not observed without cross-linking. CD45, a transmembrane tyrosine phosphatase, when co-cross-linked with either FcγRI or FcγRII, could prevent FcγRI and FcγRII-mediated calcium mobilization and protein tyrosine phosphorylation. When interleukin (IL)-6 production was measured, we noted a strong IL-6 production after activation of primary human monocytes by cross-linking of FcγRI or FcγRII. In contrast to calcium mobilization and tyrosine phosphorylation of proteins, IL-6 production was not affected by co-cross-linking of CD45 with either FcγRI or FcγRII. Interestingly, cross-linking of the CD45 itself was sufficient to induce IL-6 production. Our results show that the CD45 molecule is important in modulating early events following stimulation of primary human monocytes by cross-linking of FcγRI or FcγRII. However, triggering of CD45 alone can also induce IL-6 production, indicating that CD45 ligation itself can give signals and may have an important role in cytokine induction pathways.     

CD5− CD8αβ intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen-specific activation: CD5− and CD5+ CD8αβ IEL do not represent separate T cell lineages

We followed αβ T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I-restricted αβ TCR-transgenic (tg) mice. The proportion of tg αβ TCR⁺ CD8αβ IEL is reduced compared with CD8⁺ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8αβ IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR⁺ CD5^? CD8αβ IEL are as frequent as in specific pathogen-free (SPF) mice, whereas tg TCR⁺ CD5^int or, even more pronounced, tg TCR⁺ CD5^hi CD8αβ IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen-specific stimulation of CD5^? CD8αβ IEL in vitro, CD5 surface expression is up-regulated on a large fraction of cells within 48 h. Up-regulation of CD5 surface expression is further enhanced by the presence of the anti-αIEL monoclonal antibody 2E7. This clearly demonstrates that CD5^?, and CD5⁺ CD8αβ IEL cannot be considered as separate T cell lineages.