Characterization and Localization of the Neonatal Fc Receptor in Adult Human Kidney

J Am Soc Nephrol 11: 632–639, 2000 Characterization and Localization of the Neonatal Fc Receptor in Adult Human Kidney JEAN-PHILIPPE HAYMANN,* JEAN-PIERRE LEVRAUD,† SANDRINE BOUET,* VINCENT KAPPES,* JACQUELINE HAGEGE,*` GENEVIEVE` NGUYEN,* YICHUN XU,* ERIC RONDEAU,* and JEAN-DANIEL SRAER* *Service de Ne´phrologie A, Assistance Publique-Hoˆpitaux de Paris, Institut National de la Sante´etdela Recherche Me´dicale U489 et Association Claude Bernard, Hoˆpital Tenon, and † Institut National de la Sante´ et de la Recherche Me´dicale U277, Institut Pasteur, Paris, France. Abstract. The binding of Fc fragments of Ig on glomerular by flow cytometry, reverse transcription-PCR, Western blot- epithelial cells (GEC) was observed previously, but the recep- ting, and by the pH dependence of the binding of heat-aggre- tor could not be identified. In immunofluorescence and immu- gated IgG. Because it is well established that the FcRn is nohistochemical studies using normal adult human kidney sec- involved in IgG transcytosis, it is hypothesized that the FcRn in tions, the presence of the so-called neonatal Fc receptor (FcRn) the kidney may play a role in the reabsorption of IgG. Ongoing was demonstrated on GEC as well as in the brush border of studies should clarify the role of the FcRn as a potential target proximal tubular cells. FcRn transcripts were also detected on for immune complexes on GEC and should assess its relevance isolated glomeruli by reverse transcription-PCR. Using an im- in physiology and pathology. mortalized GEC line, the presence of the FcRn was confirmed Immune complex deposition is encountered in many glomer- transcribed in many adult tissues (7,8,19), has been identified ulonephritides. In membranous nephropathy, those deposits as the “IgG protection” receptor hypothesized by Brambell et occur between the glomerular basement membrane and vis- al. (20) to explain the paradoxically long half-life of IgG, ceral glomerular epithelial cells (GEC). This observation has relative to the half-lives of other plasma proteins. This has led led investigators to search for a specific IgG receptor on the to the hypothesis that FcRn is expressed on endothelial cells surface of GEC. It was reported that soluble aggregated IgG (21–23). To date, the expression of FcRn on extracellular Ј (AgIgG) or Fc fragments, but not F(ab )2 fragments, could bind plasma membranes has been reported only for enterocytes and to GEC in culture (1) and in tissue sections (2). However, no hepatocytes, whereas FcRn appears to be localized exclusively receptors responsible for this binding were identified, and in the cytoplasm, associated with acidified endosomes, in syn- subsequent searches for the known Fc␥ receptors CD16, CD32, cytiotrophoblast cells and some endothelial cells (13,23). and CD64 on GEC yielded negative results (3–6). FcRn mRNA has been detected by Northern blot analysis of Another Fc␥ receptor, known as the neonatal Fc receptor human kidneys (8), but the precise localization of this receptor (FcRn), has been cloned from rodents (7) and human subjects on the different structures of the kidney has not been investi- (8). This receptor is an MHC class I-like membrane protein gated. Therefore, we raised the question of whether the binding ␤ associated with 2-microglobulin. FcRn is considered to be of AgIgG or Fc fragments to GEC may be linked to the involved in IgG transport from the blood of the mother to that presence of this receptor. We show here that this receptor is of the fetus during pregnancy (8–13) and from the milk of the expressed on GEC in vivo and in vitro, as well as on the brush mother to the neonate during lactation (14–16). The ability of border of proximal tubular cells. These data raise interesting this receptor to bind IgG with higher affinity at the acidic pH questions regarding the relevance of this receptor in physio- encountered in the gut lumen, compared with the neutral logic processes and in some glomerular diseases, such as plasma pH, is thought to be important in the latter function membranous nephropathy. (16–18). However, FcRn function is not restricted to the trans- fer of IgG from mother to offspring. Indeed, FcRn, which is Materials and Methods Reagents Received February 10, 1998. Accepted September 1, 1999. The rabbit anti-FcRn antibody was a polyclonal antiserum specific Correspondence to Dr. Jean-Philippe Haymann, Service de Ne´phrologie A, for the rat FcRn heavy chain (kindly provided by Dr. Pamela Bjork- Hoˆpital Tenon, 4 Rue de la Chine, 75020 Paris, France. Phone: ϩ3315601 man, California Institute of Technology, Pasadena, CA) (18). Recog- 65 10; Fax: ϩ33 1 56 01 79 68; E-mail: [email protected] nition of the human FcRn by anti-rat FcRn antibodies was reported paris.fr previously (13). Normal rabbit serum was used as the negative con- 1046-6673/1104-0632 trol. The soluble FcRn was described previously (24) and was also Journal of the American Society of Nephrology kindly provided by Dr. Pamela Bjorkman. AgIgG was obtained by Copyright © 2000 by the American Society of Nephrology heating at 63°C for 30 min, as described (1). Anti-CD16, -CD32, and J Am Soc Nephrol 11: 632–639, 2000 FcRn Expression in Human Kidney 633 Ј -CD64 antibodies, goat anti-human F(ab )2, and human Fc fragments volume. Forty rounds of amplification, each consisting of 30 s at were purchased from Jackson ImmunoResearch (West Grove, PA). 94°C, 30 s at 60°C, and 30 s at 72°C, were then performed in a 9600 Ј FITC-conjugated anti-rabbit IgG, anti-human F(ab )2, and anti-mouse GeneAmp thermocycler (Perkin Elmer, Foster City, CA). IgG were obtained from Amersham (Orsay, France). The following oligonucleotides (purchased from Eurogentec) were used, yielding an expected 369-bp product from cDNA: FcRn1, Cell Culture 5Ј-CAAAGCTTTGGGGGGAAAAG-3Ј (hybridizing in the ␣1 do- Ј Ј Human GEC were isolated from normal tissue obtained from main); FcRn2, 5 -TGCAGGTAAGCACGGAAAAG-3 (hybridizing ␣ nephrectomies and were characterized as described previously (25). in the 3 domain). Sequencing of the PCR product was performed The cells were cultured in RMPI 1640 (Life Technologies) containing with the ABI Prism dye terminator reaction kit (Perkin Elmer), using 10% heat-inactivated fetal calf serum and 2 mM L-glutamine, and they the recommended protocol; the product was analyzed using a 373A were used between passages 3 and 4. A stable GEC cell line, E56 automated DNA sequencer (Applied Biosystems, Foster City, CA). 10A1 (hereafter referred to as E56), with a phenotype similar to that of primary cultures of GEC and podocytes in vivo (25,26) was used Immunoblot Analysis between passages 60 and 80. A human choriocarcinoma cell line Cell membranes from E56 cells and isolated glomeruli (obtained (BEWO) was obtained from the European Collection of Animal Cell after sieving) were prepared as described previously (29). Briefly, the Cultures (no. 86082803) at passage 196 and was cultured in Ham’s cells were rapidly washed three times with cold Krebs-Henseleit F-12 medium (Life Technologies) containing 2 mM glutamine and buffer (118 mM NaCl, 5 mM KCl, 1.1 mM MgSO4, 2.5 mM CaCl2, 10% fetal calf serum. 1.2 mM KH2PO4, 25 mM NaHCO3, pH 7.4) and scraped into homog- enization buffer (5 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose, Immunofluorescence Study 500 U/ml Trasylol (Bayer Pharma, Puteaux, France), 1 mM ethylene Normal portions of noninvolved poles from three tumor nephrec- glycol-bis(␤-aminoethyl ether)-N,N,NЈ,NЈ-tetra-acetic acid, and 1 mM tomy specimens were studied. The tissues were rapidly frozen in phenylmethylsulfonyl fluoride). The cells were homogenized at 0°C liquid nitrogen, and 2-␮m-thick cryostat sections were fixed in 4% in a Teflon Potter homogenizer. Two milliliters of the homogenate paraformaldehyde for 10 min and washed in phosphate-buffered sa- were loaded on 1 ml of 20 mM Tris-HCl, pH 7.5, containing 1.45 M line (PBS). The sections were incubated with the rabbit antiserum to sucrose. After centrifugation at 35,000 ϫ g for 30 min, the membranes FcRn (at a dilution of 1:40) for 30 min at room temperature, washed at the interface were collected, pelleted at 40,000 ϫ g for 20 min, and extensively with PBS, and incubated with FITC-anti-rabbit IgG for 30 washed in 10 mM Hepes, pH 7.5, containing 0.2 mM CaCl2,5mM min. Double staining was performed using a monoclonal antibody to MgCl2, 250 U/ml Trasylol, and 0.5 mM phenylmethylsulfonyl fluo- CD31 (dilution 1:100; Dako, Glostrup, Denmark), an endothelial cell ride. The cell membranes were extracted in 5% sodium dodecyl marker, and a Texas red-labeled anti-mouse IgG (Vector Laboratories, sulfate. Protein concentrations in the extracts were determined by the Burlingame, CA) for detection. The slides were then washed and method of Peterson (30). The extracts and recombinant rat FcRn were photographs were taken using immunofluorescence microscopy. resolved on 12% polyacrylamide denaturing gels and transferred to nylon membranes. The membranes were blocked with 5% nonfat milk Immunohistochemical Study in PBS and probed with rabbit antiserum to FcRn (dilution 1:500) or nonimmune control serum (dilution 1:500) overnight at 4°C. Unbound The rabbit polyclonal anti-FcRn was detected using the biotin- antibodies were removed by washing in PBS with 0.05% (vol/vol) avidin-peroxidase-coupled technique. In brief, the tissue sections were Tween 20. A second antibody, alkaline phosphatase-conjugated goat blocked with 10% normal human serum before incubation with the anti-rabbit IgG, was then applied for 30 min at 37°C, and the unbound specific rabbit polyclonal anti-FcRn antibody (dilution 1:320) for 1 h antibody was removed by washing as described above.

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