Clark L. Anderson

The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan.

The inverse relationship between serum albumin concentration and its half-life suggested to early workers that albumin would be protected from a catabolic fate by a receptor-mediated mechanism much like that proposed for IgG. We show here that albumin binds FcRn in a pH dependent fashion, that the lifespan of albumin is shortened in FcRn-deficient mice, and that the plasma albumin concentration of FcRn-deficient mice is less than half that of wild-type mice. These results affirm the hypothesis that the major histocompatibility complex–related Fc receptor protects albumin from degradation just as it does IgG, prolonging the half-lives of both.

Biology of human immunoglobulin G Fc receptors.

A heterogeneous group of receptors binding the Fc region of Ig, Fc receptors, provides important links between the cellular and humoral branches ofthe immune system. The members of this receptor group, specific for essentially all the Ig isotypes, are expressed on a variety of cells and mediate multiple important functions. Receptors for IgG (Fc’yR) , a subgroup within the larger group of FcR, were recently shown to belong to the Ig supergene family . Three major classes of leukocyte Fc R are currently recognized, all of which contain several members. Although rapid progress has been made in elucidating the structure of Fc’yR and organization of their genes [see 107] , many questions about the functions of these Fc’yR remain unanswered. In this review we focus on the various biological functions mediated by the different human Fc’yR. We utilize a new nomenclature for these receptors recently proposed by other workers (Kinet, J.-P. and Ravetch, J. , personal communication).

The MHC Class I-Like IgG Receptor Controls Perinatal IgG Transport, IgG Homeostasis, and Fate of IgG-Fc-Coupled Drugs.

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t1/2 compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.

Regulated Expression and Inhibitory Function of FcγRIIb in Human Monocytic Cells

Human monocytes/macrophages express three classes of receptors for IgG: FcγRI, FcγRII, and FcγRIII. The expression and function of these receptors has been extensively studied with the exception of one, FcγRIIb. While the mRNA for FcγRIIb has been detected in human monocytes, the protein has remained elusive. Studies in mouse models indicated that the macrophage FcγRIIb serves to down-regulate FcγR-mediated phagocytosis and immune complex-induced inflammation. FcγRIIb has also been shown to modulate the action of cytotoxic antibodies against tumors in mouse models. Hence, an understanding of how FcγRIIb expression is regulated is of great importance. Here we demonstrate for the first time FcγRIIb protein expression and function in human monocytes. We also report that the expression of FcγRIIb is highly up-regulated by interleukin-4, a Th2 cytokine, and that the up-regulation of FcγRIIb results in a decrease in the phagocytic efficiency of interleukin-4-treated THP-1 cells. Furthermore co-clustering FcγRIIb with FcγRIIa resulted in enhanced phosphorylation of the inositol phosphatase SHIP, association of SHIP with Shc, and phosphorylation of additional proteins around 120 and 60–65 kDa, with a concomitant attenuation of Akt activation. We, therefore, propose that FcγRIIb serves to inhibit FcγRI/IIa-mediated macrophage activation using SHIP as its effector.

Ethnic variation in frequency of an allelic polymorphism of human Fcγ RIIA determined with allele specific oligonucleotide probes

We have genotyped 53 individuals from three ethnic groups (Japanese, Chinese, Asian Indian) for an allotypic polymorphism of a widely expressed low affinity Fc receptor for IgG (Fc gamma RIIA). The method, requiring PCR amplification of genomic DNA and Southern analysis with allele specific oligonucleotide probes, detects a single nucleotide difference (G or A) at base 494 which results in an arginine (R) or histidine (H) at amino acid 131 of the Fc gamma RIIA protein. This polymorphism has been shown to determine the affinity of the receptor for hIgG2; Fc gamma RIIA-H131 has a high affinity for hIgG2, while Fc gamma RIIA-R131 binds hIgG2 weakly. We found that the Japanese and Chinese groups have an increased frequency of the H/H131 allotype (61 and 50% respectively) as compared to the Caucasian group (23%), in agreement with previously reported phenotype data. The genotype distribution of the Asian Indian group was not different from our Caucasian group. The shifts in frequency of the R131 and H131 alleles in different populations may have implications for disease susceptibility when the hIgG2 antibody isotype predominates.

The Fc Receptor for IgG Expressed in the Villus Endothelium of Human Placenta Is FcγRIIb2

To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we studied Fcγ receptor (FcγR) expression by immunohistology and immunoblotting. Several pan-FcγRII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical FcγRIIa were not expressed in transport-related endothelium of the placenta. Our inference, that FcγRIIb was the likely receptor, we confirmed by analyzing purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of FcγRIIb. These experiments showed that the FcγRII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly positioned to transport IgG although as well it may scavenge immune complexes.

A Novel FcγR-Defined, IgG-Containing Organelle in Placental Endothelium

Placental transfer of IgG from maternal circulation to that of the fetus is crucial for fetal and newborn immunity. This process requires that IgG broach two cellular layers of the placenta. IgG transport across the first layer, the syncytiotrophoblast, is almost certainly mediated by the MHC-related FcR for IgG, FcRn. The second layer, the villus endothelium, was until recently thought to allow IgG movement nonspecifically by constitutive transcytosis in caveolae. However, we recently showed that villus endothelium expressed a separate FcR for IgG, the inhibitory motif-bearing FcγRIIb2 seen most notably on macrophages and as a minor fraction of the FcγRIIb expressed on B cells. Now, by quantitative microscopy, we find FcγRIIb2 to be expressed abundantly in an unidentifiable and likely novel organelle of the villus endothelium, unassociated with caveolae. About half of these FcγRIIb2 organelles contain IgG; the remainder lack IgG. The majority fraction (∼80%) of IgG-containing organelles is associated with FcγRIIb. No IgG-containing organelles are associated with caveolin. These findings are compatible with FcγRIIb-mediated transfer of IgG across the villus endothelium, independent of caveolae.

Rapid and Efficient Clearance of Blood-borne Virus by Liver Sinusoidal Endothelium

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.

FcRn in the Yolk Sac Endoderm of Mouse Is Required for IgG Transport to Fetus

In adults, the nonclassical MHC class I molecule, FcRn, binds both IgG and albumin and rescues both from a degradative fate, endowing both proteins with high plasma concentrations. FcRn also transports IgG from mother to young during gestation. Anticipating that a detailed understanding of gestational IgG transport in the mouse may give us a useful model to understand FcRn function in the human placenta, we have studied FcRn in the mouse yolk sac placenta in detail. Analyzing day 19–20 fetuses of the three FcRn genotypes resulting from matings of FcRn+/− parents, we found that FcRn−/− fetuses showed negligible IgG concentrations (1.5 μg/ml), whereas IgG concentrations in FcRn+/− fetuses were about a half (176 μg/ml) that of FcRn+/+ fetuses (336 μg/ml), indicating that FcRn is responsible for virtually all IgG transport from mother to fetus. Immunofluorescence and immunoblotting studies indicated that FcRn is expressed in the endoderm of the yolk sac placenta but not in other cells of the yolk sac placenta or in the chorioallantoic placenta. IgG was found in the endoderm of both FcRn+/+ and FcRn−/− yolk sac placentas and in the mesenchyme of FcRn+/+ but was missing from the mesenchyme of FcRn−/− yolk sac placentas, indicating that IgG enters the endoderm constitutively but is moved out of the endoderm by FcRn. The similarities of these results to human placental FcRn expression and function are striking.

Src Homology 2 Domain-Containing Inositol Polyphosphate Phosphatase Regulates NF-κB-Mediated Gene Transcription by Phagocytic FcγRs in Human Myeloid Cells

FcγR-mediated phagocytosis is accompanied by the generation of tissue-damaging products such as inflammatory cytokines and reactive oxygen species. Hence, the phagocytic response must be a tightly regulated process. Recent studies have established that clustering FcγR on human myeloid cells causes tyrosine phosphorylation of Src homology 2 domain-containing inositol polyphosphate phosphatase (SHIP). However, it is not known how these immunoreceptor tyrosine-based activation motif (ITAM)-bearing phagocytic FcγR activate SHIP, or whether the activation of SHIP by ITAMs has any functional relevance. Experiments addressing the mechanism of SHIP association with ITAMs have been done in in vitro systems using phosphopeptides. In this study we undertook to dissect the molecular mechanism by which SHIP associates with the native ITAM-FcγR and becomes phosphorylated. In this report we provide evidence that first, SHIP is indeed phosphorylated by ITAM-FcγR, using cell systems that lack FcγRIIb expression; second, coimmunoprecipitation experiments demonstrate that SHIP associates with native ITAM-bearing FcγRIIa in vivo; and third, phosphorylation of SHIP by FcγRIIa is inhibited by overexpressing either the SHIP Src homology 2 domain or a dominant negative mutant of Shc. In contrast, SHIP phosphorylation was not inhibited by a dominant negative mutant of Grb2. We extend these observations to show that SHIP activation by ITAM-FcγR down-regulates NF-κB-induced gene transcription. These findings both provide a molecular mechanism for SHIP association with native ITAM-bearing receptors and demonstrate that SHIP association with ITAM-FcγR serves to regulate gene expression during the phagocytic process.