Elsevier

Human Immunology

Volume 62, Issue 2, February 2001, Pages 93-105
Human Immunology

Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells

https://doi.org/10.1016/S0198-8859(00)00244-5Get rights and content

Abstract

The mechanism of selective transport of the immunoglobulins G from the placental stroma to the lumen of the fetal blood vessels has not been elucidated yet. It was postulated that the specific transport as well as the regulation of IgG level in the blood, involves the MHC class I related receptor FcRn for the Fc domain of IgG. We questioned whether human placental endothelial cells (HPEC) express FcRn and, if present, whether it is in a functionally active form. The experiments were performed on cultured HPEC and as positive control, human trophoblastic (JEG3) and mouse endothelial cells (SVEC) were used. Expression of FcRn, was demonstrated by indirect immunofluorescence and RT-PCR. The role of FcRn was assessed by quantifying the transcellular transport of [125I]-hIgG or [125I]-rF(ab′)2 fragments from the apical to basolateral surface, and in the reverse direction of HPEC grown on filters in a double chamber system. The intracellular pathway of FcRn or IgG was examined by electron microscopy using the proteins adsorbed to 5 nm and 20 nm colloidal gold particles, respectively. The results showed that: (a) FcRn is expressed by human placental endothelial cells, in a functionally active form; (b) transcytosis of IgG in HPEC is a time-dependent process that takes place preferentially from the basolateral to the apical compartment; and (c) both IgG and FcRn colocalize in an intracellular endocytic compartment, chloroquine sensitive. Together these data suggest that the regulation of IgG level by endothelial cells may result from interplay between salvaging, exocytosis, and transcytosis of the molecules. One can assume that IgG that does not bind to FcRn may be destined for destruction, and this would explain the mechanism by which IgG homeostasis is maintained.

Introduction

One of the many functions of endothelial cells (EC) is that of providing an intelligent semipermeable barrier for transcytosis and delivery of macromolecules such as hormones, growth factors, and immunoglobulins at accurate concentration to the correct destination. For this, the EC are endowed with a dynamic system of plasmalemmal vesicles (caveolae) that may perform a highly regulated bidirectional exchange of macromolecules between blood and subendothelial tissues. It was reported that immunoglobulins G (IgG) are transported in an intact form across the placenta so as to confer passive immunity to the fetus 1, 2, 3, 4; however, the mechanism of the maternal IgG transfer through the two barriers of the placenta—the syncytiotrophoblast and the fetal capillary endothelium—is unknown.

The IgG transfer from mother to young is highly selective and is thought to involve specific receptors that bind to the Fc region of the IgG molecule, namely the MHC class I related receptor, FcRn (n from neonate) 5, 6. The vectorial transcytosis of IgG in cultured trophoblast cells was demonstrated in 1991 [7], but the involvement of FcRn in this process was shown only later when it was identified, isolated, and cloned from human placenta trophoblast cells 1, 3, 8, 9, 10. Few and contradictory data were reported about the presence of FcRn in endothelial cells. Placenta sections stained immunohistochemically with anti-hFcRn antibodies showed very low [10], occasional [11], or no expression [8] of hFcRn in fetal endothelium. Other studies reported the expression of FcRn in functionally active form in mouse endothelial cells lines 12, 13 located within vesicular structures in the cytosol. FcRn was also reported to be present in endothelial cells of human muscle vasculature [4]. Accumulated data suggests that in addition to the role of transfer of IgG from mother to young, a new function of the MHC class I related receptor, FcRn as a regulator of serum IgG homeostasis was attributed 14, 15, 16, 17. Expression of FcRn α chain mRNA not only in tissues involved in maternofetal transfer of IgG (brush border, yolk sac, and placenta), but also in other tissues of adult animals 1, 9, 12, 18, suggested that EC may be implicated in the homeostasis of IgG in the serum.

Transcytosis of macromolecules through a specific epithelial layer requires an appropriate cell culture model. Trophoblast cells in culture allowed the observation on the role of vesicles in transcytosis of IgG 7, 19; more data on the other intracellular organelles involved are needed. We took advantage of the successful isolation of endothelial cells from human term placenta (HPEC) [20] and used them in culture: (a) to search for the presence of FcRn; (b) to evaluate the binding, internalization, and transcytosis of IgG in HPEC cultured in a double chamber system; and (c) to identify by electron microscopy the intracellular compartments involved in the transcellular routing of FcRn and human IgG (hIgG). The data presented yield evidence that FcRn is expressed in a functionally active form in HPEC.

Section snippets

Chemicals

Chemicals were obtained from the following sources: MCDB131, fetal calf serum (FCS), and RNA Microisolation kit from Gibco Laboratories (Gaithersburg, MD, USA); Titan One Tube RT-PCR System from Boehringer Mannheim (Wien, Austria); Sequence T7 kit, biotinilated donkey anti-rabbit F(ab′) fragments, Streptavidin-Texas Red conjugate, and Cy3 dye from Amersham International Inc. (Buckinghamshire, England); pCR Blunt Vector from Invitrogen (Leek, Netherlands); NUCLEOTRAP Machery-Nalgen kit (Duren,

Characterization of cultured human placenta endothelial cells

Recently, a pure line of endothelial cells originating from the vasculature of human term placenta, featuring good proliferation and preserving the in vivo characteristics was obtained [20]. HPEC grown on semipermeable Trans-well membrane formed a uniform, cohesive monolayer with two distinct surface domains separated by well-organized intercellular junctions. The cells had a typical morphology exhibiting a slight elongated shape and preserve the biochemical characteristics of EC [20]. The

Discussion

It has been previously shown that FcRn plays a dual role: in the transmission of IgGs from mother to newborn and in the homeostasis of IgGs in the circulation 14, 15, 32, 33. The intracellular routing of FcRn was reported in BeWo choriocarcinoma cell line endogenously expressing FcRn and in MDCK cells expressing wild-type and mutant receptors 34, 35. No convincing data are available on the presence, localization and the role of FcRn in human placental endothelial cells, one of the main barrier

Acknowledgements

We thank Dr. Sally E. Ward (University of Texas Southwestern Medical Center at Dallas) for critical reading of the manuscript and for the suggestions made. The excellent technical assistance of F. Georgescu (radioassay), F. Chiritescu (biochemical techniques), I. Manolescu (cell culture), and N. Dobre and E. Constantinescu (photography) is gratefully acknowledged. The work was supported by a grant from the Romanian Academy and the Romanian Ministry of Research and Technology.

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