Localization of the sheep FcRn in the mammary gland

Veterinary Immunology and Immunopathology 87 (2002) 327±330

Localization of the sheep FcRn in the mammary gland BalaÂzs Mayera, Anna Zolnaia, LaÂszlo V. FrenyoÂa, Veronika Jancsikb, ZoltaÂn Szentirmayc, Lennart HammarstroÈmd, Imre Kacskovõcsa,* a

Faculty of Veterinary Science, Department of Physiology and Biochemistry, Szent IstvaÂn University, IstvaÂn utca 2, Budapest H-1078, Hungary b Faculty of Veterinary Science, Department of Anatomy and Histology, Faculty of Veterinary Science, Szent IstvaÂn University, IstvaÂn utca 2, Budapest H-1078, Hungary c National Institute of Oncology, Budapest, Hungary d CBT Novum, Karolinska Institute, Huddinge, Sweden

Abstract Among the multiple functions, which have been identi®ed for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from a pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by in situ hybridization (ISH). We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemistry con®rmed our ISH data. The cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. The presence of the FcRn in the acinar and ductal epithelial cells and the obvious change of its distribution before and after parturition suggest that FcRn plays an important role in the IgG transport during colostrum formation. FcRn expression was also found in the lamb duodenal crypt epithelial cells, which have been previously demonstrated to secrete IgG1 in newborn ruminants, suggesting secretory role of the FcRn in ruminant epithelial cells. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Sheep; FcRn; Localization; Mammary gland; IgG; Colostrum

1. Introduction In ruminants, maternal immunity is exclusively mediated by colostral immunoglobulins. Upon ingestion of the colostrum, Igs are transported across the intestinal barrier of the neonate into its blood. Whereas, this intestinal passage appears to be somewhat non-speci®c for types of Igs, there is a high selectivity in the passage of these proteins from the * Corresponding author. Fax: ‡36-1-478-4264. E-mail address: [email protected] (I. Kacskovõcs).

maternal plasma across the mammary barrier into the colostrum, and only IgG1 is transferred in large amounts. There is a rapid drop in the concentration of all lacteal Igs immediately postpartum and the selectivity of this transfer has led to the speculation that a speci®c transport mechanism across the mammary epithelial cell barrier is involved. Preferential binding of IgG1 to bovine mammary cells was previously demonstrated (for a review, see Butler, 1999). Multiple functions have recently been identi®ed for the neonatal Fc receptor FcRn. This MHC class I-related receptor was characterized originally from

0165-2427/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 0 5 9 - 4

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B. Mayer et al. / Veterinary Immunology and Immunopathology 87 (2002) 327±330

neonatal rat intestine as playing a crucial role in the passive delivery of IgGs from mother to young (Simister and Mostov, 1989). Since then this receptor was found in other epithelial barriersÐliver, mammary gland, adult intestine and kidneyÐwhere its role in IgG transport is hypothesized. Furthermore, it was detected in vascular endothelial cells regulating serum IgG levels (for a review, see Ghetie and Ward, 2000). Since the FcRn is speculated to be involved in transporting IgG into colostrum (Cianga et al., 1999) in mice, we have recently cloned the bovine counterpart of it and demonstrated its presence in multiple tissues, including the mammary gland, by Northern analysis (Kacskovõcs et al., 2000). Although, FcRn was demonstrated to be expressed in the bovine mammary gland the precise localization is needed to be elucidated. As a preliminary study, we have collected samples from a bovine mammary gland at a local slaughter house and analyzed it by in situ hybridization (ISH). We detected the bFcRn transcripts exclusively in acinar and ductal cells. Beyond the cell speci®city, the next question we wanted to resolve was the dynamics of the FcRn expression around parturition both at mRNA and at protein level. For practical purposes, we have performed this study in sheep, since this species are closely related to the bovine. We took several biopsy samples from three independent animals around parturition (24, 10 days prepartum and 1, 5, 14 and 75 days postpartum). The presence of the FcRn was analyzed by ISH and by immunohistochemistry. 2. Materials and methods 2.1. Biopsy for histological purposes Biopsies (16 g  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 on 24, 10 days prepartum and 1, 5, 14 and 75 postpartum under local anesthesia. Samples were harvested for ISH and for immunohistochemistry into freshly made 4% paraformaldehyde (PFA). 2.2. Probe preparation for in situ hybridization A 367 bp segment of the cytoplasmic and 30 untranslated regionÐshowing the lowest homology

to MHC-I genesÐfrom a sheep FcRn cDNA clone (sh 1) was ampli®ed (primers: B7 50 -GGCGACGAGCACCACTCAC-30 , B8 50 -GATTCCCGGAGGTCWCACA-30 ) in a standard PCR as follows: initial dwell for 2 min at 94 8C, denaturation at 94 8C for 30 s, annealing temperature 60 8C for 30 s and primer extension at 72 8C for 40 s. The PCR product was separated on 1% agarose gel, cut out from the gel and puri®ed on a spin column (Supelco, Bellefonte, PA). This B7±B8 fragment was added to the digoxigenin (DIG) ``labeling'' PCR reaction at the ®nal concentration of 8 ng/ml, in which a linear PCR was performed with the antisense B8 primer (the dTTP/DIGdUTP (Boehringer Mannheim, Mannheim, Germany) ratio was set to be 1.9). 2.3. In situ hybridization The biopsies were ®xed in 4% PFA and embedded in paraf®n. Tissue samples were sectioned at 5 mm and placed on silanized slides. After deparaf®nation, the sections were digested by proteinase K (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37 8C (10 mg/ml in PBS) to help the access of the probe to the mRNA in the cytoplasma. Post®xation with 4% PFA for 10 min at 4 8C was applied in order to stop the digestion. Subsequently the specimens were washed in distilled water. The DIG-labeled probe and salmon sperm DNA were denaturated at 99 8C for 5 min and added (®nal concentrations: ssDNA: 100 mg/ml, probe: 1 ng/ml) to the hybridization solution: Hybridization: 50% deionized formamide, 0.3 M NaCl, 10 mM Tris/HCl (pH 8), 1 mM EDTA, 5 Denhardt's solution, 500 ml/ml yeast tRNA (GibcoBRL-Life Technologies), 10% PEG (MW 6000), 5 mM vanadyl ribonucleoside complex (GibcoBRL-Life Technologies). This mixture was then layered on the ®xed sections and covered with coverslip. After the starting 3±5 min denaturation step at 94 8C, the ISH was carried out overnight at 42 8C on an in situ block. Next day the coverslips were removed and the sections were washed in 2 SSC 10 min and 1 SSC 10 min at room temperature and in 0.1 SSC 20 min at 42 8C. The detection was according to the Boehringer protocol used by Southern blot with slight modi®cations applying incubation with anti-DIG (200) for an hour. Color development lasted 30 min at 25 8C. Finally, the sections were washed in distilled water, air-dried and

B. Mayer et al. / Veterinary Immunology and Immunopathology 87 (2002) 327±330

mounted with Entellan (Merck, Darmstadt, Germany) for evaluation by light microscopy. 2.4. FcRn speci®c antibody New Zealand white rabbits were used to raise an antiserum against an oligopeptideÐCLEWKEPPSMRLKARPÐrepresenting aa 173±187 (bovine residues) of the a2±a3 domains, linked to maleimide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL). This peptide represents highly conserved amino acids 173± 187 of the a2±a3 domains of the bovine FcRn, plus an N-terminal Cys for conjugation. Sera containing antiFcRn was af®nity puri®ed with SulfoLink kit (Pierce, Rockford, IL). A clone (B1) of IMCD cells transfected with cDNA encoding the bovine FcRn alpha chain (Kacskovõcs et al., 2000), and untransfected IMCD cells were extracted in 1% SDS. Protein extracts were resolved on gradient polyacrylamide denaturing Tris±glycine gels based on standard protocol. Blots were probed with af®nity puri®ed anti-FcRn peptide antibody, and bound antibody was detected with horseradishperoxidase-conjugated goat anti-rabbit antibody and

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enhanced chemiluminescence (renaissance chemiluminescence reagent; NEN Life Science Products, Boston, MA). 2.5. Immunohistochemistry Sections from biopsies were prepared as previously described by ISH and placed in 1% H2O2 for 15 min to inactivate endogenous peroxidases. After a wash in distilled water 2 10 min and Tris-buffered saline (TBS) 10 min blocking was done in TBS containing 5% bovine serum albumin (BSA) for an hour. Sections were incubated with af®nity puri®ed anti-FcRn (2 dilution) in 1% BSA at 4 8C overnight and for 1 h at room temperature and then with biotinylated goat anti-rabbit IgG for 30 min at room temperature. Between each steps, slides were washed in TBS for 3 10 min. Second antibody was detected with Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and color developed using 0.25 mg/ml 3,30 -diaminobenzidine (Sigma Chemical, St. Louis, MO) in Tris buffer. Specimens were then rinsed by distilled water, air-dried and mounted with Depex.

Fig. 1. Immunohistochemical analyses of the sheep mammary gland biopsies around parturition. Strong and diffuse FcRn expression can be detected 24 and 10 days prepartum in the acinar and ductal cells. On samples derived postpartum, the FcRn appeared mainly at the apical side of these cells. Besides the different localization of this receptor, we could observe continuously weaker expression during the galactopoesis. On 75 days postpartum, diffuse localization in the cytoplasmic region appeared.

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3. Results and discussion Among the multiple functions, which have been identi®ed for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by ISH. We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemical analyses demonstrated that cytoplasm of the epithelial cells of the acini and ducti in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. Parallel with the low expression detected by the ISH, 14 days postpartum the signal was weak, nevertheless it was localized still at the apical side of the cells. Interestingly, 75 days after parturition (involution) the staining was diffuse again (Fig. 1). In ruminants, there is a special role for IgG1 in exocrine ¯uids. 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 found, demonstrating that the IgG1 was detected on the apical side if the crypt cells (Newby and Bourne, 1976). This transport contributes to the passive immune-protection of the intestinal mucosa (Besser et al., 1988). We were interested on the FcRn expression and localization on a duodenal sample derived from a newborn lamb. Based on our ®nding, this receptor was expressed by the crypt cells, and was localized dominantly to the apical side of them (Fig. 2) leading to the speculation that this receptor is involved in this transport process, as well. 4. Conclusion 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 FcRn plays an important role in the IgG transport during colostrum formation. This hypothesis is further supported by the fact that we found FcRn expression in the lamb duodenal crypt cells, which were demonstrated to secrete IgG1 in newborn calves. These data indicate that in ruminants FcRn expressed by epithelial cells which selectively bind and/or transport IgG1 into the lumen. References

Fig. 2. Immunohistochemical analysis detected FcRn at the apical side of the crypt cells on a duodenal sample derived from a neonatal lamb.

Besser, T.E., Gay, C.C., McGuire, T.C., Evermann, J.F., 1988. Passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen. J. Virol. 62, 2238±2242. Butler, J.E., 1999. Immunoglobulins and immunocytes in animal milks. In: Ogra, P.L. (Ed.), Mucosal Immunology. Academic Press, New York. Cianga, P., Medesan, C., Richardson, J.A., Ghetie, V., Ward, E.S., 1999. Identi®cation and function of neonatal Fc receptor in mammary gland of lactating mice. Eur. J. Immunol. 29, 2515±2523. Ghetie, V., Ward, E.S., 2000. Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annu. Rev. Immunol. 18, 739±766. Kacskovõcs, I., Wu, Z., Simister, N.E., Frenyo, L.V., Hammarstrom, L., 2000. Cloning and characterization of the bovine MHC class I-like Fc receptor. J. Immunol. 164, 1889±1897. Newby, T.J., Bourne, F.J., 1976. The nature of the local immune system of the bovine small intestine. Immunology 31, 475±480. Simister, N.E., Mostov, K.E., 1989. An Fc receptor structurally related to MHC class I antigens. Nature 337, 184±187.