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. 2002 Nov;107(3):288–296. doi: 10.1046/j.1365-2567.2002.01514.x

Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs

Balázs Mayer *, Anna Zolnai *, László V Frenyó *, Veronika Jancsik †, Zoltán Szentirmay ‡, Lennart Hammarström §, Imre Kacskovics *
PMCID: PMC1782797  PMID: 12423304

Abstract

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy-chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non-specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.

Introduction

The transfer of passive immunity in ruminants involves uptake of immunoglobulins from colostrum. There is a high selectivity in the transport of immunoglobulins from the maternal plasma across the mammary barrier into the colostrum, and only immunoglobulin G (IgG)1 is transferred in large amounts (reviewed in ref. 1). Upon ingestion of the colostrum, the immunoglobulins are transported across the intestinal barrier of the neonate into its blood. This intestinal passage appears to be non-specific and, subsequently, a large proportion of the absorbed IgG1 has been suggested to be recycled back into the intestinal lumen.2,3 This transport through the crypt epithelial cells2 may contribute to the protection of the gastrointestinal tract against infection during early life.4,5 The transport appears to be specific for IgG1, which, like immunoglobulin A (IgA), is relatively resistant to proteolysis.6

The transport receptor for maternal IgG in human, mouse and rat, the neonatal Fc receptor (FcRn), consists of a heterodimer of an integral membrane glycoprotein, similar to the major histocompatibility complex (MHC) class I α-chains and β2-microglobulin.7 The FcRn was first identified in rodents as the receptor that transfers maternal IgG molecules from the mother to the newborn via the neonatal intestine.8 Since then, this receptor has been detected in epithelial cells, which deliver IgG across these barriers, as well as in endothelial cells, which are responsible for the maintenance of serum IgG levels (reviewed in ref. 9).

One of several functions described for the FcRn is the regulation of IgG isotype transport into milk. Cianga and colleagues analysed the function of the mouse FcRn in the mammary gland of lactating mice. They localized the receptor to the epithelial cells of the acini and found that the transport of the IgG subclasses into milk showed an inverse correlation with their affinity to the FcRn, suggesting that the FcRn in the lactating mammary gland plays a role in recycling, rather than secreting, selected IgG subclasses from the milk gland back into the circulation.10 In the marsupial opossum, the expression of β2-microglobulin was shown to be increased when milk IgG concentration was also increased, while the expression of the α-chain was reduced after colostrum formation. In the bovine and murine mammary gland, the expression of the α-chain was constant throughout lactation, while a correlation between β2-microglobulin mRNA expression with the time of active IgG transfer into milk was also observed.11

The FcRn was originally identified in the brush border of the proximal small intestine in neonatal rodents and described as the transport receptor responsible for carrying IgG from colostrum into the blood.7,8 Although, in rodents, expression of the FcRn in intestinal epithelial cells is limited to the suckling period,12 the human receptor has been detected in both fetal and adult intestinal epithelial cells.13 While the FcRn transports IgG unidirectionally into the bloodstream in neonatal rodents, the FcRn-specific IgG transport in the human adult intestine may, based on in vitro experiments, be bidirectional, suggesting a hitherto largely neglected secretion system for host defence at the mucosal surface.14

Although FcRn has been shown to be expressed in the bovine mammary gland, its precise localization was not investigated.15 Preliminary findings in the sheep mammary gland have suggested the expression of this gene in acinar cells in a time-related manner.16 In the present work, we describe the expression and localization of the FcRn in the mammary gland in pregnant ewes around the time of parturition and also in the small intestine of the newborn lamb. Our data indicate that FcRn is involved in IgG1 secretion and thus may play an important role in mediating mucosal immunity.

Materials and methods

Cloning of the sheep FcRn heavy chain

Total RNA was isolated from sheep liver (collected at a local slaughterhouse) by using TRIzol Reagent (Gibco BRL-Life Technologies Inc., Gaithersburg, MD). Five micrograms of total RNA was reverse transcribed by using Superscript II (Gibco BRL-Life Technologies, Inc.) with the (dT)17-adapter primer (5′-GACTCGAGTCGACATCGA(T)17-3′). Based on the bovine FcRn α-chain sequence and the expected strong homology, the sheep FcRn α-chain was amplified using a primer pair that was previously used to clone the bovine FcRn in our laboratory (B10: 5′-CTGGGGCCGCAGAGGGAAGG-3′; B4: 5′-GGCTCCTTCCACTCCAGGTT-3′).15 The resultant cDNA was also subjected to 3′-rapid amplification of cDNA ends (RACE)–PCR amplification using the adapter primer (5′-GACTCGAGTCGACATCG-3′) and an FcRn-specific primer (B3: 5′-CGCAGCARTAYCTGASCTACAA-3′).

The fragments were cloned into vector pGEM-T (Promega, Madison, WI) and sequenced using an automated fluorescent sequencer from the Cybergene Company (Huddinge, Sweden).

Samples for histological examination

Biopsies (16 gauge × 16-cm length biopsy needle; Magnum, Bard, Covington, GA) were collected from the mammary gland (length of sample notch: 1·9 cm) of three ewes 24 and 10 days prepartum, and 1, 5, 14 and 75 days postpartum, under local anaesthesia, as described previously.17 To prevent local infection, 3 ml of Shotapen was injected (Virbac Laboratories, Carros, France) every 3 days throughout the experiment. Samples were harvested for in situ hybridization (ISH) and immunohistochemistry into freshly made 4% paraformaldehyde (PFA).

A duodenal sample was taken from a newborn lamb immediately after it was killed by intravenous (i.v.) administration of pentobarbital sodium injection (Nembutal; Sanofi Phylaxia, Budapest, Hungary). The sample was harvested for immunohistochemistry into freshly made 4% PFA.

All experimental procedures were approved by the Animal Care and Ethics Committee of the Faculty of Veterinary Science, Szent István University (Ref: 23/B/2000) and complied with the Hungarian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Digoxin-labelled probe preparation

A 367-bp segment of the cytoplasmic and 3′ untranslated region (showing the lowest homology to MHC-I genes) from a sheep FcRn cDNA clone was amplified (primers: B7, 5′-GGCGACGAGCACCACTCAC-3′; B8, 5′-GATTCCCGGAGGTCWCACA-3′) in a standard PCR, as follows: initial incubation for 2 min at 94°, denaturation at 94° for 30 seconds, annealing at 60° for 30 seconds, and primer extension at 72° for 40 seconds. The PCR product was separated on a 1% agarose gel, cut out from the gel and purified on a spin column (Supelco, Bellefonte, PA). This B7-B8 fragment was added to the labelling PCR to achieve a final concentration of ≈8 ng/µl. For the digoxigenin (DIG) labelling reaction, a 1·9 ratio of dTTP/DIG-dUTP (Boehringer Mannheim, Mannheim, Germany) was set and a linear PCR with the antisense B8 primer was carried out as described in a standard protocol.18 To confirm our labelling procedure, 1 µl of the probe was run on a 1% agarose gel, blotted to a Biodyne B nylon membrane (Pall BioSupport Co., East Hills, NY), UV fixed and detection carried out by using the DIG Nucleic Acid Detection kit (Boehringer Mannheim), according to the manufacturer's instructions. The colour was developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate solution for 3–12 hr in the dark.

ISH

The biopsies were fixed in 4% PFA and embedded in paraffin. Tissue samples were sectioned (5 µm) and placed onto silanized slides. After deparaffination, the sections were digested with proteinase K (Boehringer Mannheim) for 30 min at 37° (10 µg/ml in phosphate-buffered saline) to help the probe access the mRNA in the cytoplasm. Postfixation with 4% PFA for 10 min at 4° was applied in order to stop the digestion. Subsequently, the specimens were washed in distilled water. The DIG-labelled probe and salmon-sperm DNA were denatured at 99° for 5 min and added (final concentrations: salmon-sperm DNA, 100 µg/ml; probe, 1 ng/µl) to the hybridization solution, hybridization mix A,19 with some modifications [50% deionized formamide, 0·3 m NaCl, 10 mm Tris–HCl (pH 8·0), 1 mm EDTA, 5× Denhardt's solution, 500 µl/ml yeast tRNA (GibcoBRL-Life Technologies Inc.), 10% polyethylene glycol (PEG; MW 6000), 5 mm Vanadyl Ribonucleoside Complex (GibcoBRL-Life Technologies Inc.)]. This mixture was then layered onto the fixed sections and covered with coverslips. After an initial 3–5 min of denaturation at 94°, the ISH was carried out overnight at 42° on an in situ block. The next day, the coverslips were removed and the sections washed in 2 × saline sodium citrate (SSC) for 10 min and then in 1 × SSC for 10 min at room temperature and finally in 0·1 × SSC for 20 min at 42°. The detection was performed according to the Boehringer protocol with slight modifications: in our experiment anti-DIG antibody was used at a 200-fold dilution and incubated for 1 hr. Colour development lasted for 20 min at 25°. Finally, the sections were washed in distilled water, air dried and mounted with Entellan (Merck, Darmstadt, Germany) for evaluation by light microscopy.

Preparation of a FcRn-specific antiserum

New Zealand white rabbits were immunized in order to raise an antiserum against the α-chain derived peptide CLEWKEPPSMRLKAR (Agricultural Biotechnology Company, Gödöllõ, Hungary), linked to maleimide-activated keyhole limpet haemocyanin (Pierce, Rockford, IL), according to a standard protocol.19 This peptide represents the highly conserved amino acids 173–186 of the α2 and α3 domains of the bovine and sheep FcRn, plus an N-terminal Cys for conjugation. Sera containing anti-FcRn was affinity purified with a SulfoLink kit (Pierce), according to the instructions of the manufacturer.

A clone (B1) of IMCD cells transfected with cDNA encoding the bovine FcRn heavy chain15 and untransfected IMCD cells were extracted in 1% sodium dodecyl sulphate (SDS). Protein extracts were resolved on gradient polyacrylamide-denaturing Tris–glycine gels, based on a standard protocol.19 Blots were probed with unpurified and affinity-purified anti-FcRn peptide antibody, and bound antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit antibody and enhanced chemiluminescence, using luminol-based solution as substrate.20

Immunohistochemistry

Sections from biopsies and duodenal samples were prepared, as previously described, by in situ hybridization and placed in 1% H2O2 for 15 min to inactivate endogenous peroxidases. After washing in distilled water (twice, 10 min each wash) and Tris-buffered saline (TBS) (once, for 10 min), the sections were blocked by incubation in TBS containing 5% bovine serum albumin (BSA) for 1 hr. Sections were incubated with affinity-purified anti-FcRn (final concentration 76 µg/ml) in 1% BSA at 4° overnight and for 1 hr at room temperature and then with biotinylated goat anti-rabbit IgG for 30 min at room temperature. Between each step, the slides were washed in TBS (three times, 10 min each wash). The second antibody was detected using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and colour was developed using 0·25 mg/ml 3,3′-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) in Tris buffer. The specimens were then rinsed in distilled water, air dried and mounted with Entellan (Merck).

Results

Cloning of the sheep FcRn

To isolate a fragment of the sheep FcRn, we first synthesized cDNA from RNA isolated from sheep liver, as this tissue has been previously demonstrated to express FcRn in the cow15 and the rat.21,22 PCR amplification with two bovine FcRn-specific primers annealing to the 5′ untranslated region (B10) and α2 domain (B4) yielded a DNA fragment of ≈600 bp. Based on its expected size and the Southern blot verification, this amplified cDNA was ligated into vector pGEM-T and one of the clones was sequenced. We then performed 3′-RACE, using B3 and the adapter primer, which generated a DNA fragment of ≈1·3 kbp. Several of the clones obtained were sequenced. They started in the middle of the α1 domain (exon 3) and ended with a poly (A) tail. The sequence data derived from the two amplification strategies had an overlap of 397 bp and therefore it was possible to obtain a composite cDNA sequence of 1496 bp, encompassing part of the 5′ untranslated region, the entire coding segment and the 3′ untranslated region of the ovine FcRn heavy-chain cDNA (GenBank acc. no: AF421499) (Fig. 1). The data were compared with other GenBank sequences using blast, and showed a high homology to the coding region of the bovine, human and rat FcRn cDNA (96%, 78% and 65%, respectively).

Figure 1.

Figure 1

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The nucleotide sequence and deduced amino acid sequence of the sheep neonatal Fc receptor (FcRn) α-chain. The potential ATG start is marked in bold type, while the consensus initiation site is underlined. The predicted N-terminal after signal peptide cleavage is indicated by ‘+1’ under Ala. The hydrophobic membrane-spanning segment is shown by italic characters while the polyadenylation signal ‘AATAAA’ in the 3′ untranslated region is underlined. CYT, cytoplasmic; TM transmembrane.

Figure 2 shows the deduced amino acid sequence of the sheep FcRn (oFcRn) as compared to those of the bovine15 human23 and rat.7 The full-length transcript of the oFcRn α-chain we isolated was composed of three extracellular domains (α1–α2–α3), a transmembrane region and a cytoplasmic tail. It is worth mentioning that, like its cow counterpart, the oFcRn has a short cytoplasmic domain compared with all the known species analysed to date.

Figure 2.

Figure 2

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Domain-by-domain alignment of the predicted amino acid sequences for cow (b), sheep (o), human (h) and rat (r) neonatal Fc receptor (FcRn) α-chains. The N-linked glycosylation site, which is found in all the sequences, is shown by a black triangle, while white triangles indicate additional sites in the rat sequence. The grey bar indicates the hydrophobic transmembrane region. Consensus residues are assigned based on the number of occurrences of the character in the column, emphasizing the degree of conservation. The higher the conservation in a column, the darker the background of the character.38 CYT, cytoplasmic; TM transmembrane.

The sheep FcRn contains most of the residues that are known to be involved in binding to the Fc portion in the rat.24

ISH

Based on the cDNA sequence, we constructed a digoxigenin-labelled DNA probe encoding the cytoplasmic region and part of the 3′ untranslated region of the sheep FcRn to enable detection of FcRn mRNA in the mammary gland biopsies. We used ISH to localize FcRn expression in biopsies isolated from pregnant ewes 24 and 10 days before parturition, and 1, 5 and 14 days afterwards. The FcRn heavy-chain mRNA was detected by ISH exclusively in the acinar and ductal epithelial cells before and after parturition. Control sections hybridized with a sense probe derived from FcRn resulted in a weak, diffuse, non-specific background signal (Fig. 3).

Figure 3.

Figure 3

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In situ hybridization on a typical sheep mammary gland biopsy (5 days postpartum). (a) Biopsy tissue was hybridized with an anti-sense sheep neonatal Fc receptor (FcRn)-specific polymerase chain reaction (PCR)-generated, digoxigenin (DIG)-labelled probe; (b) the biopsy was hybridized with a sense probe derived from the same fragment, as a negative control [same area as (a)]. Shown are low power ( × 40). (c) and (d) A single acinus at higher power (× 100) as positive and negative samples, respectively.

Western blot and immunohistochemistry

To analyse the antisera which we raised against an oligopeptide of the α2 and α3 domain, which was identical to the bovine and sheep FcRn molecule (aa 173–186), we used a clone of bovine FcRn-transfected IMCD cells (B1; see ref. 15). Lysates of B1 cells contained a protein of the appropriate size for the FcRn α-chain (≈ 40 kDa), detected by the anti-FcRn antiserum on Western blots. No FcRn α-chain was detected in untransfected IMCD cells. Affinity purification eliminated all non-specific antibodies and resulted in a reagent, highly specific for FcRn (Fig. 4) which was used for the immunohistochemical analyses.

Figure 4.

Figure 4

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Western blot analysis of the neonatal Fc receptor (FcRn) specific antibody against an oligopeptide (CLEWKEPPSMRLKAR representing amino acids 173–186 of the α2 and α3 domains). (a) Sera, 500× dilution; (b) affinity-purified sera, 50× dilution. A clone (B1) of IMCD cells transfected with cDNA encoding the bovine FcRn heavy chain15 and untransfected IMCD cells were extracted in 1% sodium dodecyl sulphate (SDS). Arrowhead indicates the bovine FcRn α-chain (≈ 40 kDa), while numbers indicate the apparent molecular weight of the Benchmark Prestained Protein Ladder (Invitrogen).

Immunohistochemistry using affinity-purified anti-FcRn rabbit sera confirmed our ISH data. Epithelial cells of the acini in the mammary gland biopsies stained positively around parturition (Fig. 5), although there was a remarkable difference in the pattern before and after parturition. Before parturition (24 and 10 days prepartum), the staining was diffuse, indicating an even distribution of the FcRn α-chain throughout the acinar epithelial cells (Fig. 5a, 5b). After parturition (1, 5 and 14 days postpartum) the apical/luminal sides of the epithelial cells were more markedly stained than the cytoplasm (Fig. 5c, 5d, 5e). We also observed a downward trend in FcRn expression, analysing the samples 14 days postpartum (Fig. 5e). These sections show a weaker signal at the apical region of some epithelial cells and regions where the FcRn presence is barely visible. Consistent with the in situ hybridization, there was no staining of endothelial cells in the lamina propria or muscular tunics. At 75 days postpartum, diffuse localization reappeared in the cytoplasm (Fig. 5f).

Figure 5.

Figure 5

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Immunohistochemical analyses of the sheep mammary gland biopsies around parturition. Strong and diffuse neonatal Fc receptor (FcRn) expression was detected 24 (a) and 10 days (b) prepartum in the acinar and ductal cells. On samples derived postpartum day 1 (c), day 5 (d) and day 14 (e), the FcRn appeared mainly at the apical side of these cells. At 75 days postpartum (f), diffuse localization was observed in the cytoplasmic region. L, lumen of the acini. The main panels are shown at low power ( × 40) and the inserts at higher power ( × 100).

We also investigated the FcRn expression and localization on a duodenal sample derived from the newborn lamb. Using the affinity-purified FcRn-specific antiserum, a marked staining was seen in the apical portions of crypt epithelial cells. We also saw weaker staining at the basal side and a scattered staining in the cytoplasm of these cells, whereas there was no staining in the enterocytes (Fig. 6). The scattered staining in the lamina propria may be caused by the staining of intestinal macrophages, which in humans have been shown to express FcRn.25

Figure 6.

Figure 6

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Immunohistochemical analysis detected strong apical and weak basal (arrows) neonatal Fc receptor (FcRn) in the duodenal crypt cells of a neonatal lamb. However, FcRn was not detected in the duodenal enterocytes. Magnification, × 20.

Discussion

Although IgG1 and IgG2 are present at approximately equal concentrations in ruminant blood, only the IgG1 subclass is transported from blood across the alveolar epithelial cell into the mammary secretions.26 The alveolar transportation of IgG1 is intensified about 2–3 weeks prior to parturition and coincides with a decrease in the IgG1 concentration of blood.27 Immunoglobulin transmission through the mammary epithelial cells has been studied in detail, and, in ruminants, maternal immunity is mediated exclusively by colostral immunoglobulins.1 The receptor responsible for the transport of IgG1 in this tissue has not yet been identified, although previous studies have indicated that specific binding sites exist on mammary epithelial cells around the time of parturition. The differential distribution of IgG1 and IgG2 has been determined in prepartum and lactating bovine mammary tissue; IgG1 was found predominantly within the alveolar epithelial cells and lumens of prepartum tissue, whereas IgG2 was largely confined to the stromal area surrounding the alveoli. Both IgG subclasses were restricted predominantly to the stroma in lactating tissue.28,29 Dispersed cells from digests of mammary tissue have also been used in binding studies and suggest preferential binding of IgG1 to mammary cells prior to calving.30

We have recently cloned the bovine FcRn and demonstrated its expression in multiple tissues (including the mammary gland and the small intestine) using Northern analysis.15 Although the FcRn was shown to be expressed in the bovine mammary gland, its precise localization was not investigated. Preliminary findings in the sheep mammary gland have suggested the expression of this gene in acinar cells in a time-related manner.16 In the present study, the FcRn heavy-chain mRNA was detected exclusively in the acinar and ductal epithelial cells in all the samples before and after parturition, with no major differences in expression (Fig. 3). Immunohistochemical analysis demonstrated that the cytoplasm of the epithelial cells of the acini and ducti in the mammary gland biopsies stained homogeneously before parturition, although a marked difference was observed in this pattern after lambing. The signal indicated uneven distribution of the FcRn α-chain in the epithelial cells 1 and 5 days postpartum, because the apical sides of the epithelial cells were stained most strongly. By 14 days postpartum, the signal was weak, but nevertheless still localized at the apical marginal side of the cells (Fig. 5).

Based on these data, we hypothesize that the FcRn in ruminant mammary gland selectively binds IgG1 at the basal side of the acinar epithelial cells and transports it to the luminal side, providing IgG1 in the colostrum and, subsequently (although to a much lesser extent), in the milk. This hypothesis is, however, in contrast to the suggested function of the mouse FcRn in the mammary gland of lactating mice. Cianga and colleagues have localized the receptor to the epithelial cells of the acini and found that the transport of IgG subclasses into milk showed an inverse correlation with their affinity to the FcRn, indicating that the FcRn in the lactating mammary gland plays a role in recycling IgG from milk glands back into the circulation.10

In contrast to the mouse model, we hypothesize that the ruminant FcRn transports IgG1 into the lumen and is probably not involved in recycling IgG2 back to the circulation. The receptor would then have to fulfil at least two requirements: first, it should show higher affinity to IgG1 in the binding and/or the transport process; and second, it should mediate basolateral-to-apical IgG transport in these cells. As previous studies detected IgG1, but not IgG2, within the mammary acinar cells,29,30 where we detected the FcRn, we propose that the ruminant FcRn probably favours binding to IgG1. Besides this indirect evidence, we consider that analysing the affinities of IgG1 and IgG2 for the FcRn receptor would be of critical importance for resolving this argument. Current studies in our laboratory involving in vitro IgG1 and IgG2 binding and transport experiments in bovine FcRn-transfected MAC-T cells (a bovine mammary acinar-cell derived cell line; see ref. 31) are attempting to resolve this issue.

Concerning the second point, some studies on the FcRn transcytosis have indicated significant basolateral-to-apical transport.14,32 Additionally, the cytoplasmic region of the ruminant FcRn molecules is shorter by 10 amino acids than their rodent and human counterparts (Fig. 2). One may speculate that the lack of this segment may lead to a significant shift to the basolateral-to-apical transport in ruminants; however, further studies on the cytoplasmic region are required to answer this question.

Our data also suggest that the transcription of the FcRn heavy chain is not down-regulated markedly, if at all, in association with the increased lactogenic activity and decreased secretion of IgG1. These data are in good agreement with a recent study that has found a constant level of FcRn mRNA in the bovine mammary gland throughout lactation, whereas, in contrast, an increased level of β2-microglobulin mRNA in the mammary gland correlated with the time of active IgG-transfer into milk.11 Our immunohistochemical data indicated not only a different localization but also a downward trend of the FcRn expression postpartum, suggesting that the presence of the FcRn in the mammary gland was controlled by the expression of β2-microglobulin and that its presence was up-regulated during the period of highest IgG transfer into colostrum and subsequently down-regulated. The co-expression of β2-microglobulin with the α-chain of FcRn is therefore probably required for appropriate maturation and function, as well as for stable expression in ruminants, as has been recently demonstrated in human FcRn.33,34

Upon ingestion of colostrum, the immunoglobulins are transported across the intestinal barrier of the neonate into its blood. Newborns absorb all proteins non-discriminately during the first 12 hr after birth. At 12 hr, the ability of the enterocytes to pinocytotically transport immunoglobulin rapidly declines and by 18–24 hr it is totally lost. The exact mechanism leading to ‘gut closure’ is unknown, although maturation of the lysosomal system is suspected.1 Whereas this intestinal passage appears to be non-specific, a large proportion of the absorbed IgG1 is recycled back into the small intestinal lumen in young ruminants.2,3 This transport, which is mediated by the crypt epithelial cells,2 contributes to protection of the intestinal mucosa against infections.4

The predominance of IgG1 in mucosal fluid supports the concept of a special role for IgG1 in mucosal immunity in ruminants,5 which can be explained by the fact that IgG1, similarly to IgA, is more resistant to proteolysis than IgG2.6 In an examination of the immunoglobulins of the small intestine of calves, IgG1 was the major immunoglobulin in the secretions, and IgA was present in smaller amounts. Histological evidence of transport of IgG1 across the crypt epithelial cell was previously found, demonstrating that IgG1 was detected on the apical side of the crypt cells.2 Based on our findings in a newborn lamb, the FcRn was expressed by the crypt cells and was mainly localized at the apical side (Fig. 6), leading to speculation that this receptor is involved in this transport process. We were not able to detect FcRn in enterocytes (which are responsible for the initial absorption of intestinal IgG), supporting previous findings that immunoglobulin uptake is a non-specific event.

The function of FcRn in the intestine of suckling mice and rats has been well documented.35 In rodents, the FcRn is expressed at high levels by intestinal epithelial cells and mediates absorption of IgG by receptor-mediated transcytosis. FcRn expression is developmentally down-regulated, resulting in almost complete loss of intestinal FcRn at the time of weaning.12,36,37 The FcRn, in adult human intestinal epithelial cells, has been detected by immunohistochemistry, which demonstrated strong apical staining in the apical (luminal) region.13 In a more recent study, Dickinson and colleagues demonstrated that the FcRn is expressed not only in the enterocytes but also in the crypt epithelial cells in the adult small intestine. In both cell types, the FcRn accumulated in the apical region. Dickinson et al. have also shown that FcRn mediates bidirectional IgG transport in a polarized human intestinal T84 cell line, and the basolateral-to-apical transcytosis was favoured. These data raise the possibility that the FcRn may function to transport IgG across the adult human intestinal epithelium and it may then play an important role in immunosurveillance.14

The presence of the FcRn in the acinar and ductal epithelial cells in the mammary gland, and the obvious change of its distribution before and after parturition, suggest that it plays an important role in the transport of IgG during colostrum formation. This hypothesis is further supported by the fact that FcRn expression was found in the lamb duodenal crypt epithelial cells, which were demonstrated to secrete IgG1 in newborn calves.

These data indicate that in ruminants the FcRn expressed by epithelial cells selectively binds and/or transports IgG1 into the lumen, which may contribute to local mucosal immunoprotection. In the cow, IgG1 is well represented in mucosal fluids (such as saliva and tears) and tissues (such as the small and large intestine, lung and genitourinary tract).5 The mechanism by which IgG is transported onto these mucosal surfaces is currently unknown but, based on the functions described in this report, we speculate that it is mediated by the FcRn.

Acknowledgments

We would like to thank Ágnes Méhes and Ilona Horn for excellent technical assistance with the immunohistochemistry and Ágnes Mészáros for preparing the tissue paraffin sections. This work was supported by the National Research Fund of Hungary (OTKA T035209, T030304); Research and Development Fund for Hungarian Higher Education (FKFP 0672), and the Swedish Research Council.

Abbreviations

DIG

digoxigenin

ISH

in situ hybridization

NBT/BCIP

nitroblue tetrazolium salt/5-bromo-4-chloro-3-indolyl-phosphate

PFA

paraformaldehyde

RACE

rapid amplification of cDNA ends

References

  • 1.Butler JE. Immunoglobulins and immunocytes in animal milks. In: Ogra PL, editor. Mucosal Immunology. 2. New York: Academic Press; 1999. pp. 1531–54. [Google Scholar]
  • 2.Newby TJ, Bourne FJ. The nature of the local immune system of the bovine small intestine. Immunology. 1976;31:475–80. [PMC free article] [PubMed] [Google Scholar]
  • 3.Besser TE, McGuire TC, Gay CC, Pritchett LC. Transfer of functional immunoglobulin G (IgG) antibody into the gastrointestinal tract accounts for IgG clearance in calves. J Virol. 1988;62:2234–7. doi: 10.1128/jvi.62.7.2234-2237.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Besser TE, Gay CC, McGuire TC, Evermann JF. Passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen. J Virol. 1988;62:2238–42. doi: 10.1128/jvi.62.7.2238-2242.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Butler JE. Bovine immunoglobulins: an augmented review. Vet Immunol Immunopathol. 1983;4:43–152. doi: 10.1016/0165-2427(83)90056-9. [DOI] [PubMed] [Google Scholar]
  • 6.Newby TJ, Bourne FJ. Relative resistance of bovine and porcine immunoglobulins to proteolysis. Immunol Commun. 1976;5:631–5. doi: 10.3109/08820137609033871. [DOI] [PubMed] [Google Scholar]
  • 7.Simister NE, Mostov KE. An Fc receptor structurally related to MHC class I antigens. Nature. 1989;337:184–7. doi: 10.1038/337184a0. [DOI] [PubMed] [Google Scholar]
  • 8.Rodewald R. pH-dependent binding of immunoglobulins to intestinal cells of the neonatal rat. J Cell Biol. 1976;71:666–9. doi: 10.1083/jcb.71.2.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Ghetie V, Ward ES. Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annu Rev Immunol. 2000;18:739–66. doi: 10.1146/annurev.immunol.18.1.739. [DOI] [PubMed] [Google Scholar]
  • 10.Cianga P, Medesan C, Richardson JA, Ghetie V, Ward ES. Identification and function of neonatal Fc receptor in mammary gland of lactating mice. Eur J Immunol. 1999;29:2515–23. doi: 10.1002/(SICI)1521-4141(199908)29:08<2515::AID-IMMU2515>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  • 11.Adamski FM, King AT, Demmer J. Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum) Mol Immunol. 2000;37:435–44. doi: 10.1016/s0161-5890(00)00065-1. [DOI] [PubMed] [Google Scholar]
  • 12.Martin MG, Wu SV, Walsh JH. Ontogenetic development and distribution of antibody transport and Fc receptor mRNA expression in rat intestine. Dig Dis Sci. 1997;42:1062–9. doi: 10.1023/a:1018853506830. [DOI] [PubMed] [Google Scholar]
  • 13.Israel EJ, Taylor S, Wu Z, Mizoguchi E, Blumberg RS, Bhan A, Simister NE. Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells. Immunology. 1997;92:69–74. doi: 10.1046/j.1365-2567.1997.00326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Dickinson BL, Badizadegan K, Wu Z, Ahouse JC, Zhu X, Simister NE, Blumberg RS, Lencer WI. Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest. 1999;104:903–11. doi: 10.1172/JCI6968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kacskovics I, Wu Z, Simister NE, Frenyo LV, Hammarstrom L. Cloning and characterization of the bovine MHC class I-like Fc receptor. J Immunol. 2000;164:1889–97. doi: 10.4049/jimmunol.164.4.1889. [DOI] [PubMed] [Google Scholar]
  • 16.Mayer B, Zolnai A, Frenyo LV, Jancsik V, Szentirmay Z, Hammarstrom L, Kacskovics I. Localization of the sheep FcRn in the mammary gland. Vet Immunol Immunopathol. 2002;87:327–30. doi: 10.1016/s0165-2427(02)00059-4. [DOI] [PubMed] [Google Scholar]
  • 17.Colitti M, Stradaioli G, Stefanon B. Effect of alpha-tocopherol deprivation on the involution of mammary gland in sheep. J Dairy Sci. 2000;83:345–50. doi: 10.3168/jds.S0022-0302(00)74885-5. [DOI] [PubMed] [Google Scholar]
  • 18.Wilkinson DG. New York: Oxford University Press; 1992. In Situ Hybridization. [Google Scholar]
  • 19.Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Greene Publishing Associates and Wiley-Interscience. New York: John Wiley & Sons; 1989. Current Protocols in Molecular Biology. [Google Scholar]
  • 20.Kricka LJ. Chemiluminescent and bioluminescent techniques. Clin Chem. 1991;37:1472–81. [PubMed] [Google Scholar]
  • 21.Blumberg RS, Koss T, Story CM, et al. A major histocompatibility complex class I-related Fc receptor for IgG on rat hepatocytes. J Clin Invest. 1995;95:2397–402. doi: 10.1172/JCI117934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Telleman P, Junghans RP. The role of the Brambell receptor (FcRB) in liver: protection of endocytosed immunoglobulin G (IgG) from catabolism in hepatocytes rather than transport of IgG to bile. Immunology. 2000;100:245–51. doi: 10.1046/j.1365-2567.2000.00034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Story CM, Mikulska JE, Simister NE. A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus. J Exp Med. 1994;180:2377–81. doi: 10.1084/jem.180.6.2377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Martin WL, West AP, Jr, Gan L, Bjorkman PJ. Crystal structure at 2.8 A of an FcRn/heterodimeric Fc complex: mechanism of pH-dependent binding. Mol Cell. 2001;7:867–77. doi: 10.1016/s1097-2765(01)00230-1. [DOI] [PubMed] [Google Scholar]
  • 25.Zhu X, Meng G, Dickinson BL, et al. MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells. J Immunol. 2001;166:3266–76. doi: 10.4049/jimmunol.166.5.3266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Larson BL, Heary HL, Jr, Devery JE. Immunoglobulin production and transport by the mammary gland. J Dairy Sci. 1980;63:665–71. doi: 10.3168/jds.S0022-0302(80)82988-2. [DOI] [PubMed] [Google Scholar]
  • 27.Sasaki M, Davis CL, Larson BL. Production and turnover of IgG1 and IgG2 immunoglobulins in the bovine around parturition. J Dairy Sci. 1976;59:2046–55. doi: 10.3168/jds.S0022-0302(76)84486-4. [DOI] [PubMed] [Google Scholar]
  • 28.Kemler R, Mossmann H, Strohmaier U, Kickhöfen B, Hammer DK. In vitro studies on the selective binding of IgG from the different species to tissue sections of the bovine mammary gland. Eur J Immunol. 1975;5:603–8. doi: 10.1002/eji.1830050905. [DOI] [PubMed] [Google Scholar]
  • 29.Leary HL, Jr, Larson BL, Nelson DR. Immunohistochemical localization of IgG1 and IgG2 in prepartum and lactating bovine mammary tissue. Vet Immunol Immunopathol. 1982;3:509–14. doi: 10.1016/0165-2427(82)90016-2. [DOI] [PubMed] [Google Scholar]
  • 30.Sasaki M, Larson BL, Nelson DR. Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells. Biochim Biophys Acta. 1977;497:160–70. doi: 10.1016/0304-4165(77)90149-0. [DOI] [PubMed] [Google Scholar]
  • 31.Huynh HT, Robitaille G, Turner JD. Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp Cell Res. 1991;197:191–9. doi: 10.1016/0014-4827(91)90422-q. [DOI] [PubMed] [Google Scholar]
  • 32.Schlachetzki F, Zhu C, Pardridge WM. Expression of the neonatal Fc receptor (FcRn) at the blood–brain barrier. J Neurochem. 2002;81:203–6. doi: 10.1046/j.1471-4159.2002.00840.x. [DOI] [PubMed] [Google Scholar]
  • 33.Praetor A, Hunziker W. Beta(2)-microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn. J Cell Sci. 2002;115:2389–97. doi: 10.1242/jcs.115.11.2389. [DOI] [PubMed] [Google Scholar]
  • 34.Claypool SM, Dickinson BL, Yoshida M, Lencer WI, Blumberg RS. Functional reconstitution of human FcRn in MDCK cells requires co-expressed human beta (sub2}m. J Biol Chem. 2002;277:28038–50. doi: 10.1074/jbc.M202367200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Junghans RP. Finally! Brambell Receptor (FcRB) Immunol Res. 1997;16:29–57. doi: 10.1007/BF02786322. [DOI] [PubMed] [Google Scholar]
  • 36.Berryman M, Rodewald R. Beta 2-microglobulin co-distributes with the heavy chain of the intestinal IgG-Fc receptor throughout the transepithelial transport pathway of the neonatal rat. J Cell Sci. 1995;108:2347–60. doi: 10.1242/jcs.108.6.2347. [DOI] [PubMed] [Google Scholar]
  • 37.Ghetie V, Hubbard JG, Kim JK, Tsen MF, Lee Y, Ward ES. Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur J Immunol. 1996;26:690–6. doi: 10.1002/eji.1830260327. [DOI] [PubMed] [Google Scholar]
  • 38.Nicholas KB, Nicholas HBJ. GeneDoc: a tool for editing and annotating multiple sequence alignments. www.psc.edu/biomed/gendoc.

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