Colostrogenesis: Role and Mechanism of the Bovine Fc Receptor of the Neonate (FcRn).

Colostrogenesis is a separate and unique phase of mammary epithelial cell activity occurring in the weeks before parturition and rather abruptly ending after birth in the bovine. It has been the focus of research to define what controls this process and how it produces high concentrations of specific biologically active components important for the neonate. In this review we consider colostrum composition and focus upon components that appear in first milked colostrum in concentrations exceeding that in blood serum. The Fc Receptor of the Neonate (FcRn) is recognized as the major immunoglobulin G (IgG) and albumin binding protein that accounts for the proteins' long half-lives. We integrate the action of the pinocytotic (fluid phase) uptake of extracellular components and merge them with FcRn in sorting endosomes. We define and explore the means of binding, sorting, and the transcytotic delivery of IgG1 while recycling IgG2 and albumin. We consider the means of releasing the ligands from the receptor within the endosome and describe a new secretion mechanism of cargo release into colostrum without the appearance of FcRn itself in colostrum. We integrate the insulin-like growth factor family, some of which are highly concentrated bioactive components of colostrum, with the mechanisms related to FcRn endosome action. In addition to secretion, we highlight the recent findings of a role of the FcRn in phagocytosis and antigen presentation and relate its significant and abrupt change in cellular location after parturition to a role in the prevention and resistance to mastitis infections.

Quantification of Neonatal Fc Receptor and Beta-2 Microglobulin in Human Liver Tissues by Ultraperformance Liquid Chromatography-Multiple Reaction Monitoring-based Targeted Quantitative Proteomics for Applications in Biotherapeutic Physiologically-based Pharmacokinetic Models.

Neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) play an important role in transporting maternal IgG to fetuses, maintaining the homeostasis of IgG and albumin in human body, and prolonging the half-life of IgG- or albumin-based biotherapeutics. Little is known about the influence of age, gender and race, and interindividual variability of human FcRn and ß2M on the protein level. In this study, an ultraperformance liquid chromatography-multiple reaction monitoring mass spectrometry-based targeted quantitative proteomic method was developed and optimized for the quantification of human FcRn and ß2M. Among the 39 human livers studied (age 13-80 years), the mean (±S.D.) concentrations of FcRn and ß2M were 147 (±39) and 1250 (±460) pmol/g of liver tissue, respectively. A four-fold interindividual variability (63-243 pmol/g of liver tissue) was observed for the hepatic FcRn concentration. A moderate correlation was found between the hepatic ß2M and FcRn expression levels. Influences of age, gender, and race on the hepatic expression of FcRn and ß2M were evaluated. The findings from this study may aid the development of physiologically-based pharmacokinetic models that incorporate empirical FcRn tissue concentrations and interindividual variabilities, and the development of personalized dosing of biopharmaceuticals. SIGNIFICANCE STATEMENT: This is the first study to evaluate the influence of age, gender, and race on the expression of neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) and their interindividual variability in human livers. This study describes a validated ultraperformance liquid chromatography-multiple reaction monitoring-based targeted quantitative proteomic method for quantifying human FcRn and ß2M in biological tissues. Results from this study may aid current development of physiologically-based pharmacokinetic models for biotherapeutics, where FcRn plays a significant role in clearance mechanism, and its expression level and interindividual variability are largely unknown.

Quantitative Analysis of Human Neonatal Fc Receptor (FcRn) Tissue Expression in Transgenic Mice by Online Peptide Immuno-Affinity LC-HRMS.

Neonatal Fc receptor (FcRn) is the homeostatic receptor responsible for the long half-life of endogenous IgG by protecting it from lysosomal degradation. Understanding systemic FcRn tissue expression is important to predict and design the half-life of therapeutic antibodies and Fc-coupled biotherapeutics. To this end, we measured human FcRn (hFcRn) tissue expression in Tg32, a human FcRn knock-in transgenic mouse model, for which a strong correlation of drug clearance to humans has been demonstrated. Building an hFcRn tissue expression profile in Tg32 was enabled by the development of a tissue preparation procedure composed of bead-based protein extraction and protein precipitation using acetone followed by pellet digestion with trypsin. Digests were then loaded onto an online peptide immuno-affinity flow configuration hyphenated with reversed phase nanoflow chromatography and coupled with high resolution mass spectrometry to quantify hFcRn derived peptides. The workflow allowed bypassing some of the challenges typically associated with membrane protein analysis. We demonstrated acceptable precision and bias for measuring hFcRn in tissue matrices, typically within 20% coefficient of variation and relative error. We also report hFcRn expression in several Tg32 tissues. We anticipate that establishing a quantitative approach for hFcRn in tissues will enable the systematic measurement of hFcRn concentrations to further increase the accuracy of physiologically based pharmacokinetic (PBPK) models for PK prediction of Fc-containing biotherapeutics. This is anticipated to improve the translation of pharmacokinetic data from preclinical model systems to humans.

FcRn Expression on Placenta and Fetal Jejunum during Early, Mid-, and Late Gestation in Minipigs.

Developmental toxicity testing of therapeutic antibodies is most often conducted in nonhuman primates owing to lack of cross-reactivity in other species. Minipigs may show cross-reactivity for some humanized antibodies but have not been used for developmental toxicity testing due to an assumed lack of embryo-fetal exposure. Unlike in humans, maternal IgGs do not cross the porcine placenta to reach the fetus. Some humanized IgGs, however, have a higher affinity for the neonatal Fc receptor (FcRn) and are more likely than endogenous antibodies to cross the placenta of animals. The major site of prenatal IgG transfer is the placenta, though FcRn in fetal intestine could also uptake maternal IgGs from swallowed amniotic fluid. Using immunohistochemistry andin situhybridization in this experiment, FcRn was found in minipig placenta and fetal intestine during early, mid-, and late gestation. To date, however, fetal exposure to maternally administered IgGs has never been demonstrated in the minipig.

Skewed B cells in chronic hepatitis C virus infection maintain their ability to respond to virus-induced activation.

Chronic hepatitis C virus (HCV) infection is characterized by persistent B-cell activation, with enhanced differentiation and reduced proliferative ability. To assess the possible role of HCV in altering B-cell subset distribution, we examined ex vivo frequencies and B-cell inhibitory receptor expression in 37 chronic HCV-infected patients and 25 healthy donors (HD). In addition, we determined whether short-term exposure to culture-derived HCV (HCVcc) resulted in B-cell subset skewing and/or activation. There was a statistically significant increase in the frequencies of immature transitional, activated memory and tissue-like memory (TLM) B cells in HCV-infected patients compared with HD. We also found that the frequency of memory B cells correlated with serum HCV RNA levels. The proportion of B cells expressing the marker of exhaustion Fc receptor-like 4 (FcRL4) was generally low even though significantly higher in the patients' memory B-cell compartment compared with HD, and a positive correlation was found between the frequencies of the patients' TLM FcRL4+ B cells and serum alanine aminotransferase and histological activity index at liver biopsy. Exposure to cell-free HCVcc in vitro did not result in B-cell skewing but induced significant activation of naïve, TLM and resting memory B cells in HCV-infected patients but not in HD, in whom cell-associated virus was an absolute requirement for activation of memory B cells. These findings provide corroborative evidence in favour of significant B-cell subset skewing in chronic HCV infection and in addition show that expression of exhaustion markers in selected B-cell subsets does not impair virus-induced B-cell activation.

Emerging roles for the FCRL family members in lymphocyte biology and disease.

Members of the extended Fc receptor-like (FCRL) family in humans and mice are preferentially expressed by B cells and possess tyrosine-based immunoregulatory function. Although the majority of these proteins repress B cell receptor-mediated activation, there is an emerging evidence for their bifunctionality and capacity to counter-regulate adaptive and innate signaling pathways. In light of these findings, the recent discovery of ligands for several of these molecules has begun to reveal exciting potential for them in normal lymphocyte biology and is launching a new phase of FCRL investigation. Importantly, these fundamental developments are also setting the stage for defining their altered roles in the pathogenesis of a growing number of immune-mediated diseases. Here we review recent advances in the FCRL field and highlight the significance of these intriguing receptors in normal and perturbed immunobiology.

The old but new IgM Fc receptor (FcµR).

IgM is the first Ig isotype to appear during phylogeny, ontogeny and the immune response. The importance of both pre-immune "natural" and antigen-induced "immune" IgM antibodies in immune responses to pathogens and self-antigens has been established by studies of mutant mice deficient in IgM secretion. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed, but fail to fully account for the IgM-mediated immune protection and regulation of immune responses. Particularly, the role of the Fc receptor for IgM (FcµR) in such effector functions has not been explored until recently. We have identified an authentic FcµR in humans using a functional cloning strategy and subsequently in mice by RT-PCR and describe here its salient features and the immunological consequences of FcµR deficiency in mice. Since the FcµR we cloned was identical to Toso or Fas inhibitory molecule 3 (FAIM3), there have been spirited debates regarding the real function of FcµR/Toso/FAIM3 and we will also comment on this topic.

Nodal reactive and neoplastic proliferation of monocytoid and marginal zone B cells: an immunoarchitectural and molecular study highlighting the relevance of IRTA1 and T-bet as positive markers.

AIMS: Marginal zone B cells (MZCs) and monocytoid B cells (MBCs) appear to be related lymphoid cells that take part in reactive and neoplastic marginal zone proliferations. These lesions are not yet well characterized, and the aim of this study was to find better diagnostic criteria for them. METHODS AND RESULTS: We analysed 60 nodal lesions with MBC and/or MZC proliferation for their morphological, immunophenotypic, molecular genetic and IG gene rearrangement features. On the basis of the results of the rearrangement assay and immunoglobulin light chain restriction, the lesions were divided into reactive and neoplastic groups. Among the neoplastic lesions, polymorphic and monomorphic subgroups emerged. All reactive lesions had morphological features of the polymorphic subgroup. By immunohistochemistry, IRTA1 and/or T-bet expression was found in all reactive lesions and in 90% of neoplastic lesions. CONCLUSIONS: IRTA1 and T-bet are positive markers for the identification of MZC/MBC proliferations, and thus for the diagnosis of nodal marginal zone lymphoma (NMZL). Polymorphic and monomorphic subgroups of NMZL could be distinguished. Most morphological and immunophenotypic patterns in reactive and neoplastic nodal expansions of MZCs and MBCs overlapped. Therefore, PCR clonality assay of the immunoglobulin heavy and light chain gene loci is the most reliable method for their differentiation.

Methods for Functional Characterization of FcRn Interactions with Therapeutic Antibodies and Fc-Fusion Proteins.

The neonatal Fc receptor (FcRn) plays a key role in determining the pharmacokinetic behavior of therapeutic monoclonal antibodies (mAbs). FcRn-mediated intracellular trafficking mechanisms extend the half-lives of mAbs by rescuing them from lysosomal degradation and contribute to their transportation from the vascular space to tissue compartments such as placenta and mucosal surfaces. It is important to characterize the FcRn interactions of therapeutic mAbs and Fc-fusion proteins due to its potential impact on their in vivo pharmacokinetic properties such as clearance and half-life. In this chapter, we describe protocols for two cell-based assays that measure the total function of FcRn which involves pH-dependent association and dissociation with IgG-Fc, as well as FcRn-mediated intracellular trafficking parameters. These assays are suitable for characterization of FcRn interactions with therapeutic mAbs and Fc-fusion proteins for the purpose of assessing lot-to-lot consistency and the structural and functional integrity of the Fc domain. In addition, they may serve as cost-effective screening tools for the evaluation of mAb-based drug candidates during lead selection and optimization for desired pharmacokinetic properties.

Atypical memory B cells are greatly expanded in individuals living in a malaria-endemic area.

Epidemiological observations in malaria endemic areas have long suggested a deficiency in the generation and maintenance of B cell memory to Plasmodium falciparum (Pf) in individuals chronically reinfected with the parasite. Recently, a functionally and phenotypically distinct population of FCRL4(+) hyporesponsive memory B cells (MBCs) was reported to be expanded in HIV-infected individuals with high viral loads. In this study, we provide evidence that a phenotypically similar atypical MBC population is significantly expanded in Pf-exposed Malian adults and children as young as 2 years of age as compared with healthy U.S. adult controls. The number of these atypical MBCs was higher in children with chronic asymptomatic Pf infections compared with uninfected children, suggesting that the chronic presence of the parasite may drive expansion of these distinct MBCs. This is the first description of an atypical MBC phenotype associated with malaria. Understanding the origin and function of these MBCs could be important in informing the design of malaria vaccines.

Best practices for optimization and validation of flow cytometry-based receptor occupancy assays.

In the development of therapeutic compounds that bind cell surface molecules, it is critical to demonstrate the extent to which the drug engages its target. For cell-associated targets, flow cytometry is well-suited to monitor drug-to-target engagement through receptor occupancy assays (ROA). The technology allows for the identification of specific cell subsets within heterogeneous populations and the detection of nonabundant cellular antigens. There are numerous challenges in the design, development, and implementation of robust ROA. Among the most difficult challenges are situations where there is receptor modulation or when the target-antigen is expressed at low levels. When the therapeutic molecules are bi-specific and bind multiple targets, these challenges are increased. This manuscript discusses the challenges and proposes best practices for designing, optimizing, and validating ROA.

The potential of porcine ex vivo platform for intestinal permeability screening of FcRn-targeted drugs.

Conventionally, the intestinal permeability of drugs is evaluated using cell monolayer models that lack morphological, physiological and architectural features, as well as realistic neonatal Fc receptor (FcRn) expression. In addition, it is time-consuming, expensive and excessive to use a large number of mice for large-scale screening of FcRn-targeted candidates. For preclinical validation, it is critical to use suitable models that mimic the human intestine; the porcine ex vivo model is widely used for intestinal permeability studies, due to its physiological and anatomical similarities to humans. This study intended to analyze the potential to measure the intestinal permeability of FcRn-targeted substances using a porcine ex vivo platform, which is able to analyze 96 samples at the same time. In addition, the platform allows the screening of FcRn-targeting substances for transmucosal delivery, taking into consideration (cross-species) receptor-ligand binding kinetics. After analyzing the morphology of the porcine tissue, the FcRn expression across the gastrointestinal tract was verified. By studying the stomach, duodenum and jejunum, it was demonstrated that FcRn expression is maintained for up to 7 days. When evaluating the duodenum permeability of free engineered human albumin variants, it was shown that the variant with the mutation K573P (KP) is more efficiently transported. Given this, the porcine ex vivo platform was revealed to be a potential model for the screening of FcRn-targeted oral drug formulations.

Macrophage activation selectively enhances expression of Fc receptors for IgG2a.

After infection with bacillus Calmette-Guérin, peritoneal macrophages (Mø) display enhanced expression of FcR for both monomeric and complexed IgG2a, but not IgG2b. Isotype specificity of FcR can be reversed on nonactivated Mø by immune lymphokines, and IgG2a immune complexes are more effective triggers of the respiratory burst in activated Mø. Selective enhancement of IgG2a FcR by Mø activation could account for efficacy of homologous ab in mediating cytotoxicity in some systems.

Phenotypic characterization of human cytolytic T lymphocytes in mixed lymphocyte culture.

Mixed lymphocyte reaction (MLR)-activated T cells were analyzed according to the expression of various cell surface markers by the specific cytotoxic T lymphocytes (CTL) generated in the MLR. CTL were found exclusively in a population of MLR-activated T cells that lacked detectable Fc gamma R but that expressed a surface antigen recognized by the 4F2 monoclonal antibody. In contrast, CTL were found in both the Ia-positive and Ia-negative cells after MLR activation. Thus, the specific CTL generated in the allogeneic MLR can be identified and isolated by virtue of the expression of a particular cell surface marker.

Biochemical nature of an Fc mu receptor on human B-lineage cells.

An IgM-binding protein of approximately 60 kD has been identified on activated B cells, but not on resting and activated T cells, monocytes, or granulocytes. Here, we characterize this IgM-binding protein as a receptor for the Fc portion (CH3 and/or CH4 domains) of IgM molecules (Fc microR). The Fc microR can be expressed as a cell surface activation antigen throughout the pre-B and B cell stages in differentiation. Receptor expression is not directly linked with IgM production, as both mu- pre-B cells and isotype-switched B cells may express the Fc microR. The receptor molecules produced by both pre-B and B cells are identical in size and are characterized as an acidic sialoglycoprotein with O-linked, but no N-linked, oligosaccharide. The Fc microR is anchored to the surface of B-lineage cells via a glycosyl phosphatidylinositol linkage. The Fc microR is thus the third member of a family of Fc receptors expressed on B-lineage cells, and its preferential expression on activated B cells suggests a potential role in the response to antigens.

Pre-B cells and other possible precursor lymphoid cell lines derived from patients with X-linked agammaglobulinemia.

A group of unique Epstein-Barr virus-containing cell lines was derived from the bone marrow of three patients with X-linked agammaglobulinemia. Efforts to obtain cell lines from the peripheral blood of these patients were uniformly unsuccessful. Immunofluorescence analyses as well as biosynthetic studies with [(35)S]methionine indicated unusual patterns of Ig synthesis in many of these bone marrow derived lines. Seven of the lines were of particular interest in that two produced no Ig of any type; two others showed no Ig by fluorescence but small amounts by [(35)S]methionine labeling; one expressed only cytoplasmic mu chains without any evidence of light chain synthesis, and two produced primarily mu chains with only slight amounts of light chains. One of the lines without membrane or cytoplasmic Ig studied in detail grew like a typical lymphoid line and was carried in intermittent culture over a period of 2 yr without Ig expression. One line grew quite differently and resembled the round cell type described previously, which has been obtained from a variety of sources. The cell line with cytoplasmic mu chains and no light-chain expression had the characteristic properties of pre-B cells. Three normal type Ig-producing cell lines also were obtained from the patients. The accumulated evidence obtained in the present study indicates that these unusual cell lines represent normal precursor cells of the B-cell lineage; these grew out in these cases because of the virtual absence of mature B cells that ordinarily overgrow the culture system. However, the possibility that in certain instances they reflect abnormal Ig synthesis characteristic of the disease has not been ruled out.

Interleukin 2 and stimulator lymphoblastoid cells will induce human thymocytes to bind and kill K562 targets.

Human thymocytes cultured in the presence of IL-2 and an irradiated B cell line became cytotoxic to K562 target cells. Thymocytes cultured alone or with only IL-2 exhibited almost no killing, but thymocytes cultured in the presence of stimulator cells alone exhibited low levels of cytotoxic activity. Removal of Fc gamma receptor-bearing cells from the activated thymocyte population almost completely abolished the binding and lytic activity. Separation of thymocytes into Fc microns+ and Fc microns-cells before culturing with IL-2 and stimulator cells revealed that only the Fc microns+ subpopulation developed into K562 killer cells. These findings indicate that modulation of Fc microns to Fc gamma receptors on the thymocyte cell surface is part of the maturation process of this particular subset of cytotoxic cells. Morphologically, most of the activated Fc gamma+ K562-binding cells were large, granulated lymphocytes. Only very few of the round, nongranulated small thymocytes were bound to K562 target cells.

In vitro differentiation of human monocytes. Differences in monocyte phenotypes induced by cultivation on glass or on collagen.

We demonstrated that the in vitro differentiation of human peripheral blood monocytes to macrophages is dependent on the environment and conditions of monocyte culture. Cultivation of monocytes on glass or microexudate-coated glass gave rise to cells resembling foreign body granuloma macrophages. After an initial rise in Fc receptor- and C3 receptor-mediated phagocytosis, a progressive loss of Fc receptor expression and C3-mediated ingestion were observed. The monocyte surface antigens recognized by the anti-human monocyte monoclonal antibodies 1D5 and 63D3 were lost from the surface of the majority of cells cultured on glass and microexudates. A subpopulation of Fc receptor-positive cells that were 1D5 and 63D3 positive was retained in fully differentiated cell populations. In comparison, monocytes cultivated on collagen matrices gave rise to highly phagocytic cells resembling human resident tissue macrophages. Both Fc- and C3-mediated phagocytosis were enhanced and remained so during the entire length of culture. The surface antigens recognized by the 1D5 antibody, expressed on all freshly seeded monocytes, was maintained on the macrophages. The antigen recognized by the 63D3 antibody was not expressed on mature cells. The present evidence would indicate that variations in expression of phagocytic receptors and the surface antigens 1D5 and 63D3 can be ascribed to the stage of development of the macrophage or its stage of activation, rather than to independent subsets of mononuclear phagocytes.

Receptor-mediated immunoglobulin G transport across mucosal barriers in adult life: functional expression of FcRn in the mammalian lung.

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain the immunoglobulins (Ig)G and secretory IgA (sIgA) that function together in host defense. Exactly how IgG crosses epithelial barriers to function in mucosal immunity remains unknown. Here, we test the idea that the MHC class I-related Fc-receptor, FcRn, transports IgG across the mucosal surface of the human and mouse lung from lumen to serosa. We find that bronchial epithelial cells of the human, nonhuman primate, and mouse, express FcRn in adult-life, and demonstrate FcRn-dependent absorption of a bioactive Fc-fusion protein across the respiratory epithelium of the mouse in vivo. Thus, IgG, like dimeric IgA, can cross epithelial barriers by receptor-mediated transcytosis in adult animals. These data show that mucosal surfaces that express FcRn reabsorb IgG and explain a mechanism by which IgG may act in immune surveillance to retrieve lumenal antigens for processing in the lamina propria or systemically.

Developmentally regulated association of a 56-kD member of the surface immunoglobulin M receptor complex.

Immature and mature B cells differ in the signals generated and transduced through their antigen receptor, surface immunoglobulin M (sIgM). Whereas signals generated through sIgM on mature B cells initiate a program leading to the positive activation of these cells, signaling through this receptor at the immature stage of development leads to a state of induced unresponsiveness or tolerance. Our previous studies have described developmental differences in sIgM transmembrane signaling that are independent of ligand-receptor affinity. In an attempt to understand the molecular basis for signaling differences between immature and mature B cells, we have analyzed the sIgM receptor complex in neonatal and adult mouse splenic B cells. While previously described components of this complex do not exhibit marked developmentally regulated differences in their association with sIgM, we have identified a 56-kD protein that associates with sIgM in mature (antigen-responsive), but not immature (tolerance-sensitive) B cells. This protein (p56) associates with sIgM as a homodimer, is constitutively phosphorylated on tyrosine, and is coimmunoprecipitated with IgM but not IgD. The observed inability to iodinate p56 suggests it is an intracellular component of the receptor complex. Based upon its migration in one- and two-dimensional gel electrophoresis we show, however, that p56 is distinct from the blk, lyn, or fyn src family kinases that have been shown to be associated with sIgM in mature B cells. The developmentally regulated participation of p56 in the B cell antigen receptor complex suggests a role in the differential signaling mediated via sIgM on immature and mature B cells.