Expression pattern of the human FcRH/IRTA receptors in normal tissue and in B-chronic lymphocytic leukemia

A new family of Ig domain receptors referred to as the immune receptor translocation-associated (IRTA) proteins, FcR homologs (FcRHs) or FcR-like that are expressed in lymphoid cells has been recently described. RNA expression analysis suggests that FcRH1-5/IRTA1-5 are expressed exclusively in subsets of the B-cell compartment. We generated mAbs to FcRH1-5/IRTA1-5 and examined their protein expression pattern in normal tissue and in chronic lymphocytic leukemia (CLL) cells. Our data indicated that FcRH1-5/IRTA1-5 were expressed in B-cell sub-populations; however, in some cases, the protein was not expressed in the same B-cell populations as suggested by the RNA expression analysis. FcRH1/IRTA5 was expressed throughout the B-cell lineage starting at the pro-B-cell stage but was down-regulated in plasma cells. FcRH2/IRTA4 was expressed preferentially in memory B cells. FcRH3/IRTA3 was expressed at low levels in naive, germinal center (GC) and memory B cells but was also expressed in NK cells. FcRH4/IRTA1 was expressed in a sub-population of memory B cells associated with mucosal tissue. FcRH5/IRTA2 was expressed in mature B cells and memory B cells and down-regulated in GC cells and, unlike all other B-cell-specific markers, maintained its expression in plasma cells from tonsil, spleen and bone marrow. We examined the expression of FcRH1-5/IRTA1-5 on the surface of CLL cells and found a similar pattern of expression on CLL cells as in the normal mature B cells, except for FcRH3/IRTA3 which was up-regulated in CLL.     

Fc receptor homologs: newest members of a remarkably diverse Fc receptor gene family

Summary: Newfound relatives of the classical Fc receptors (FcR) have been provisionally named the Fc receptor homologs (FcRH). The recent identification of eight human and six mouse FcRH genes substantially increases the size and functional potential of the FcR family. The extended family of FcR and FcRH genes spans ～15 Mb of the human chromosome 1q21–23 region, whereas in mice this family is split between chromosomes 1 and 3. The FcRH genes encode molecules with variable combinations of five subtypes of immunoglobulin (Ig) domains. The presence of a conserved sequence motif in one Ig domain subtype implies Ig Fc binding capability for many FcRH family members that are preferentially expressed by B lineage cells. In addition, most FcRH family members have consensus tyrosine‐based activating and inhibitory motifs in their cytoplasmic domains, while the others lack features typical of transmembrane receptors. The FcRH family members, like the classical FcRs, come in multiple isoforms and allelic variations. The unique individual and polymorphic properties of the FcR/FcRH members indicate a remarkably diverse Fc receptor gene family with immunoregulatory function.     

Differential B cell expression of mouse Fc receptor homologs

Five Fc receptor homologs (FcRH1-5) possessing inhibitory and/or activating signaling motifs are differentially expressed during B cell differentiation in humans. In this analysis we describe their three mouse orthologs, moFcRH1, moFcRH2 and moFcRH3. The moFcRH genes are located in a chromosome 3 region that is syntenic with the FcRH locus on human chromosome 1. They encode proteins with 2-5 Ig-like domains that share 20-61% extracellular identity with their human counterparts. One moFcRH1 isoform lacks a transmembrane domain as do both moFcRH2 isoforms. The other moFcRH1 isoform and two moFcRH3 isoforms have transmembrane domains and cytoplasmic ITIM and ITAM-like consensus sequences implying their inhibitory or activating signaling potential. Whereas the moFcRH1 and moFcRH3 orthologs are preferentially expressed at different stages in B cell differentiation, the structurally novel moFcRH2 gene is expressed in non-lymphoid tissues. The highly restricted pattern of moFcRH3 expression suggests this member of the phylogenetically conserved FcRH family may have an important immunoregulatory role in marginal zone B cells.     

Fcγ receptors contribute to pyramidal cell death in the mouse hippocampus following local kainic acid injection

Recent studies have demonstrated the contribution of the gamma subunit of the Fc receptor of IgG (FcRγ) to neuronal death following ischemic injury and Parkinson's disease. We examined the role of FcRγ in hippocampal pyramidal cell death induced by kainic acid (KA). FcRγ-deficient mice (FcRγ−/−) and their FcRγ+/+ littermates (wild type, B6) received an injection of KA into the dorsal hippocampus. Pyramidal cell death was quantified 24 and 72 h after the injection. The number of survived pyramidal cells was significantly larger in FcRγ−/− mice than in B6 mice in both the CA1 and CA3. Immunohistochemical and immunofluorescent studies detected FcγRIIB protein in parvalbumin neurons, whereas FcγRIII and FcγRI proteins were detected in microglial cells. No activated microglial cells were detected 24 h after the KA injection in FcRγ−/− mice, whereas many activated microglial cells were present in B6 mice. The production of nitrotyrosine as well as of the inducible nitric oxide synthase and cyclooxygenase-2 proteins, increased by 16 h after the KA injection in B6 mice. In addition, tissue plasminogen activator and metalloproteinase-2 proteins increased. By contrast, the magnitude of oxidative stress and the increase in protease expression were mild in FcRγ−/− mice. Co-injection of a neutralizing antibody against FcγRll and FcγRlll with KA abolished pyramidal cell death and microglial activation. In addition, the neutralizing antibody reduced oxidative stress and expression of proteases. These observations suggested a role for FcγRllB in parvalbumin neurons as well as FcRγ in microglia in pyramidal cell death.     

Macrophage function in high- and low-responder strains of mice

Unstimulated peritoneal macrophages of the high-responder A/J mice compared to the low-responder B10 strain have a lower number of cells with the different Fc receptor (FcR) subtypes and correspondingly the FcR mediated phagocytosis is also lower. The intraperitoneal immunization with SRBC leads to a prompt increase of FcR expression and of phagocytic and pinocytic activity in the A/J mice, while in the B10 strain the activity of macrophages remains unchanged.     

Size-dependent effect of IgA on the IgA Fc receptor (CD89)

The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat-aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR γ chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence-activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 ± 2% positive cells. Incubation of the cells with different sizes of IgA-containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1- or IgA2-containing aggregates. Furthermore, the binding of aIgA was found to be CD89-specific, since the binding of IgA was completely inhibited by the CD89-specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin-2 (IL-2) production as a marker, showed that the FcR γ chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR γ chain (CD89⁺/γ⁺) enhance the IL-2 production 10–12-fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca²⁺]_i) in cells co-expressing FcR γ chain. Mutation of the tyrosine residues in the FcR γ chain immunoreceptor tyrosine-based activation motif of the FcR γ chain abolishes this increase in [Ca²⁺]_i, indicating association and involvement of the FcR γ chain in CD89-mediated signaling.     

抗Fc受体γ链单克隆抗体的研制及生物学特性鉴定

目的:制备Fc受体γ链的单克隆抗体(mAb),并对其生物学特性进行鉴定,为研究受体γ链相关的疾病提供实验材料。方法:人工合成的蛋白多肽偶联后免疫BALB/c小鼠,用间接ELISA法筛选阳性克隆,Western blot、流式细胞术(FCM)检测鉴定抗体的特性及抗原识别位点。结果:通过细胞融合和亚克隆,共筛选出3株能稳定分泌抗体并且反应性好的杂交瘤细胞株5B6、7D3和8D4。其中7D3抗体反应性最强,2.5μg/mL与体内体外的γ链都能特异性反应。结论:获得了抗Fc受体γ链特异性的mAb,为进一步研究Fc受体γ链在相关疾病发生发展过程中所发挥的作用提供了良好的研究手段。     

FcR cross-linking on monocytes results in impaired T cell stimulatory capacity

Presentation of antigen to T lymphocytes without the appropriateco-stlmulatory signals results in a state of antigen-specificunresponsiveness. Despite the presumed importance of the B7-CD28interaction for the initiation and maintenance of T cell-mediatedimmune responses, relatively few studies have addressed theregulation of B7 expression. We have studied the expressionof the CD80 (B7-1) and B7-2 molecules on peripheral blood monocytesfollowing different activation signals, and it was demonstratedthat not only IFN-, but also granulocyte macrophage colony stimulatingfactor can induce CD80 expression on monocytes. In addition,we found that cross linking of FcR on monocytes strongly inhibitsthe up-regulation of CD80 and B7-2, with as a functional consequencethat the capacity to function as antigen presenting cells (APC)and to.stimulate T cell activation is severely impaired. Whencultures were prepared In 96-well plates coated with human IgG,stimulation of T cells with allogenelc monocytes resulted onlyin modest T cell proliferation and no detectable IL-2 secretionas compared with untreated culture plates or plates coated withFab fragments of human IgG. Under these conditions cross-linkingof CD28 on the T cells with specific mAb completely revertedthe inhibitory effect observed after culture on IgG-coated plates.Furthermore, FcR cross-linking on monocytes strongly Inhibitedthe capacity of monocytes to Induce a specific memory T cellresponse to viral, bacterial and fungal antigens, whereas thetreatment did not impair the capacity of the T cells to respondto pokeweed mitogen, phytohemagglutinin and concanavalin A.We conclude that after FcR cross-linking, the impaired APC functionis most likely due to the inability of monocytes to providethe essential costimulatory signals to the T cells via the B7–CD28/CTLA-4interaction.     

A striking example of convergent evolution observed for the ggFcR:IgY interaction closely resembling that of mammalian FcR:IgG

We have recently identified a novel IgY specific chicken FcR (ggFcR) on chromosome 20, a region where no FcR gene is present in mammals. Serially deleted IgY fusion proteins were tested in a reporter assay to identify C_H domains involved in ggFcR binding. Single C_H domains did not bind to ggFcR, whereas Fcυ2 to Fcυ4 induced good and the Fcυ3 to Fcυ4 domains moderate activity. When IgY from diverse birds were assayed, only IgY from gallinaceous birds showed binding, which enabled us to pinpoint several potential contact sites by a sequence comparison and molecular modelling. Point mutations of critical residues at these sites revealed the Fcυ2 and Fcυ3 domains as major ggFcR:IgY binding sites similar to mammalian IgG. These results demonstrate that ggFcR has a contact site to IgY which closely resembles that of human IgG bound to FcR.     

A monoclonal antibody inhibiting human placental Fc gamma-receptor activity

Fc gamma receptor (FcR) from human placenta was solubilized using EDTA and 2-mercaptoethanol and purified by affinity chromatography on human IgG-coated Sepharose 4B. BALB/C mice were immunized with FcR and monoclonal antibodies were obtained by growing hybridoma cells following fusion of spleen cells with P3 X 63Ag8 myeloma cells. Using an immunofluorescence technique, the IgG1 monoclonal antibody secreted by clone B1D6 stained the FcR-positive areas in sections of placental tissue. The endothelium of the foetal stem vessels stained more strongly than did the trophoblasts. The antibody also inhibited the haemadsorption to placental tissue of erythrocytes (E) sensitized with IgG antibodies (A), (EA), and inhibited the agglutination of EA by FcR. The data indicate that the monoclonal antibody reacts with the placental FcR at the binding site for IgG, or with an epitope close to the binding site. Apparently, the FcR in different anatomical areas in the placenta have a common antigenic determinant.     

IgG Fc receptors on epithelial cells of distal tubuli and on endothelial cells in human kidney

IgG Fc receptors (FcR) in cryostat sections of human kidney were studied using functional assays and monoclonal antibodies (MoAbs). Using functional assays, FcR were detected on cells in glomeruli and distal convoluted tubuli, on endothelial cells and on interstitial cells. These cells were also stained with B1D6, a MoAb reactive with a 40-kD molecule with low affinity FcR activity. Using MoAbs against leukocyte FcR, a different pattern of reactivity was obtained. MoAbs against FcR I (32.2), FcR II (IV.3 and C1KM5) and FcR III (3G8 and Leu11b) stained interstitial macrophages only. The data confirm the presence of FcR on renal glomeruli and interstitial cells and also indicate that epithelial cells of distal tubuli and endothelial cells express FcR. Furthermore, renal FcR appear to be structurally different from the FcR I, II and III defined on leukocytes.     

Activating and inhibitory FcgammaRs in autoimmune disorders

Autoimmune disorders are characterized by the destruction of self-tissues by the immune system. Multiple checkpoints are in place to prevent autoreactivity under normal circumstances. Coexpression of activating and inhibitory Fc receptors (FcR) represents such a checkpoint by establishing a threshold for immune cell activation. In many human autoimmune diseases, however, balanced FcR expression is disturbed. Analysis of murine model systems provides strong evidence that aberrant FcR expression can result in uncontrolled immune responses and the initiation of autoimmune disease. This review will summarize this data and explain how this information might be used to better understand human autoimmune diseases and to develop novel therapeutic strategies.     

Effect of Fc receptor blocking on human natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The effect of Fc receptor (FcR) blocking by aggregated human gamma-globulin (AGG) was studied on natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of [³H]TdR-prelabelled HEp-2 cells. LDCC was evaluated in a 24 h assay at 50:1 effector-target cell ratio in the presence of 25 μg/ml concanavalin A (Con A). Under these conditions but without Con A considerable NCMC was not elicited by normal lymphocytes. FcR blocking by AGG treatment of effector cells resulted in a significant NCMC activity to HEp-2 targets. In contrast, AGG treatment profoundly depressed LDCC. Monocyte depletion of effector cells had no major influence on the effect of AGG on NCMC and LDCC activities. An interference of FcR blocking by AGG and LDCC in response to Con A is suggested.     

Do Immune Complexes Formed with Autoantibodies Have a Role in the Maintenance of Immune Homeostasis Through Interaction with FC Receptors

Natural autoantibodies play an important regulatory role in the maintenance of immune homeostasis. They act as a first line of defense against environmental pathogens like toxins, bacteria and erythrocytes. In humans they are mainly produced by CD5+ B cells that are under the control of a regulatory T cell population. Fc-γ receptors are involved in antigen recognition and signal transduction and tuning, and some of the members of the FcR family have structural similarity to MHC molecules; they may interact with multiple Ig ligands and with non-Ig ligands.

We discuss the interactions between immune-complexes formed with natural autoantibodies and Fc-γ receptors and suggest that such interactions may affect self-recognition in the thymus and regulate immune homeostasis     

Human IgG isotypes and activating Fcγ receptors in the interaction of Salmonella enterica serovar Typhimurium with phagocytic cells

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.     

FcRγ deficiency improves survival in experimental sepsis by down-regulating TLR4 signaling pathway

Fc receptor common γ signaling chain (FcRγ), a common subunit shared by Fc receptors (FcγRI, III, IV, FcαRI, and FcεRI), is an important immune regulator both in innate and adaptive immunity. Previous studies have shown that FcRγ was a potential target of inflammatory diseases, whereas the role of FcRγ in sepsis has been poorly understood. In this study, we found that deficiency of FcRγ resulted in increased survival in lipopolysaccharide (LPS)/D-galactosamine and E. coli-induced sepsis in mice. This protective effect was characterized by decreased TNF-α, IL-6, and IL-10. Further experiments in bone marrow-derived macrophages (BMDMs) in vitro also showed that FcRγ deficiency resulted in decreased production of TNF-α, IL-6, and IL-10 upon LPS stimulation. The mechanism study showed that FcRγ was physiologically associated with toll-like receptor 4 (TLR4), and tyrosine phosphorylation of FcRγ mediated TLR4 signaling pathway, followed by increased ERK phosphorylation upon LPS stimulation. Our results suggest that FcRγ might be a potential therapeutic target of sepsis.

     

Inhibitory mechanism of the proliferative response of B lymphocytes: suppression of the proliferation induced by anti-mu antibody and BSF1 by immune complexes.

We studied the effect of immune complexes (IC) on the responses of polyclonally activated murine B cells. For this, normal resting B cells were stimulated with the F(ab')2 fraction of goat anti-mouse mu-chain antibody and B-cell stimulating factor 1 (BSF1) after preculturing them with IC. Next, the relative membrane potential changes and the subsequent proliferative response were analysed. IC, particularly in antibody excess, inhibited both membrane depolarization and while those in antigen excess did poorly. Neither antigen nor antibody alone was effective. Inhibition was mediated via binding of IC to FcR gamma on B cells in a dose-dependent manner. Kinetic experiments showed that at least 6 hr was necessary for inducing the suppression of B-cell responses after binding of IC. We conclude that IC bound to FcR gamma on B cells regulates B-cell responses by acting on the initial step of activation.     

Cross-talk between pathogen recognizing Toll-like receptors and immunoglobulin Fc receptors in immunity

The individual role of pathogen-binding Toll-like receptors (TLRs) and antibody-binding Fc receptors (FcRs) during pathogenic infections has been studied extensively. However, combined activation of these different receptor classes has received little attention, even though they are triggered simultaneously when immune cells bind antibody-opsonized pathogens. In the last few years, it has become evident that joined activation of TLRs and FcRs substantially tailors inflammatory immune responses, which is an efficient and controlled mechanism of the host to act upon invading pathogens. In this review, we discuss the mechanisms of cross-talk between different TLRs and FcRs and the resulting inflammatory immune responses. Furthermore, we propose how chronic activation via this cross-talk might be detrimental in inflammatory (auto) immune diseases. We conclude with the potential exploitation of the interplay between TLRs and FcRs for monoclonal antibody therapy to target tumors. Future interests in this field of research include establishing a more detailed and mechanistic understanding of the mode of action of TLR and FcR cross-talk and exploration of its physiological importance in health and disease. This may furthermore open up novel therapeutic options for intervention in inflammatory diseases or cancer.