Histochemistry(1992) 98 : 359 364
Histochemistry © Springer-Verlag 1992
Expression of intestinal alkaline phosphatase in human organs UHa Domar 1, Berith Nilsson 1, Vladimir Baranov 2, Ulf Gerdes 3, Torgny Stigbrand 1 1 Department of Medical Biochemistryand Biophysics,Universityof UmeS, S-90187 UmeA,Sweden 2 Department of Immunology,Universityof Ume~, S-90187 Ume~, Sweden 3 Department of Pathology,Universityof Umefi, S-90187 Umefi,Sweden Accepted: 14 September 1992
Abstract. Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.
Introduction Human alkaline phosphatases ALP; EC 22.214.171.124) comprise a group ofisozymes, which are selectively expressed in defined organs. The isozymes are attached by a phosphatidylinositol glycan anchor (PIG-tail) to the plasma membrane of specific cells. The intestinal alkaline phosphatase (IAP) appears mainly in the small intestine, bound to the brush border of the epithelial cells. The placental alkaline phosphatase (PLAP) is localized in the syncytiotrophoblasts of term placenta, and the tissue unspecific alkaline phosphatase (AP) is expressed in rainCorrespondence to: U. Domar
eralizing bone and in the liver and kidney (Fishman 1974). The ALP isozyme genes have been cloned (Berger et al. 1987; Kam et al. 1985; Millan 1986; Weiss et al. 1986), and the complete amino acid sequences are known, revealing homologies of about 87% between lAP and PLAP and 50-60% between AP and the two other isozymes (Harris 1989). Recently, several observations indicating a more complex pattern of expression of the ALP isozymes have been reported, i.e. IAP has been immunochemically identified in the liver and kidney (Hirano et al. 1987), and an IAP-like isozyme has been purified from human kidney (Hirano et al. 1989b). Trace amounts of PLAP have been found in the testis, ovary and lung (Goldstein et al. 1982; Hirano et al. 1989a), and AP seems to be expressed in almost all organs (Goldstein et al. 1982). Verpooten et al. (1989) localized an IAP-like ALP in the proximal tubules of the kidney. The immunohistochemical identification and localization of the IAP isozyme in organs beside the intestine and the kidney has to our knowledge not been previously reported. The pronounced homologies between the ALP isozymes cause immunochemical cross-reactivity, which severely hampers their selective immunohistochemical identification in organs where they are expressed together and only in trace amounts. The hybridoma technology (K6hler and Milstein 1975) has made new excellent immunochemical tools available with which to identify single epitopes on proteins, and thus made it possible to discriminate between closely related molecules. This makes selective identification of the ALP isozymes possible despite the high homology between the proteins. The aim of this study was to produce a monoclonal antibody specific for the IAP isozyme and suitable for immunohistochemical studies of the expression and localization of IAP in human organs, in which AP and PLAP might also be expressed.
Materials and methods
Isozymes of alkaline phosphatase Pure IAP was prepared from human small intestine as previously described (Domar 1987) with immuno-affinity chromatography using the anti-IAP monoclonal antibody 2HIMS-1 as the last purification step. Pure liver ALP and PLAP were prepared from human liver and placenta respectively using the techniques described by Sugiura et al. (1975) and Holmgren and Stigbrand (1976) but by use of the isozyme specific monoclonal antibodies HLMS-1 and HPMS-1 respectively in a final affinity chromatography step. All tissues were obtained by autopsy from the Dept. of Pathology and Forensic Medicine at the University Hospital, U m e L
Monoclonal antibodies Monoclonal antibodies (Mabs) were raised by fusing spleen cells from BALB/c mice immunized with purified IAP, with cells of the SP2/0 myeloma cell line. The mice were immunized intraperitoneally with 50 gg of purified IAP in Freund's complete adjuvant, and several booster injections of 20 to 100 gg of pure IAP in Freund's incomplete adjuvant were given. When the mice had reached a high serum titre of anti-IAP antibodies, booster injections of 100 gg of partially purified IAP were given intravenously on 3 consecutive days. On the fourth day the spleens were removed and prepared for fusion as described by Uotila et al. (1980). The identification of specific antibodies from the growing clones after HAT (hypoxantine-aminopterine-thymidine) selection was done by enzyme-linked immunosorbent assay (ELISA; Engvall 1980) with pure IAP as the coating antigen. Positive hybridomas were expanded, cloned and grown as ascitic tumours in Pristane-primed BALB/c mice.
were prepared free floating in cacodylate buffer. Blocking of endogenous peroxidase, restoration of the three-dimensional structure of the protein and blocking of unspecific binding sites were performed by incubation of the sections with, respectively, 5 mM periodic acid in 0.01 M PBS, pH 7.2 for 10 min, 3 mM boric hydride (NaBH~) in PBS for 20 min, and 0.2% bovine serum albumin in PBS for about 20 min at room temperature. The sections were then incubated for 2 h at room temperature with a solution of the monoclonal antibody IAP4 in tissue culture supernatant diluted 1:2 with PBS and including 0.05% saponin. Between all the incubations the tissue sections were extensively washed with PBS. Finally the second antibody, F(ab)2-fragments of anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP), was incubated overnight at 8° C, followed by washing and then with the substrate for HRP (0.05 M diaminobenzidine and 0.003% H202) for 20 min. All tissue sections were washed three times in 0.05 M TRIS buffer, pH 7.6, and then re-fixed in a 1% solution of osmium tetroxide, buffered in 0.1 M phosphate buffer at pH 7.2 for 1 h, dehydrated and embedded for conventional electron microscopy.
Processing of tissues for light microscopy Normal tissues from human pancreas, kidney, liver, gall bladder and bile duct were obtained at surgery from the Department of Pathology, University Hospital, Uppsala. The tissues were routinely formalin-fixed and embedded in paraffin blocks. Sections 5 gm in thickness were cut and mounted on poly-L-lysine coated slides (Sigma), and the slides were deparaffinized, rehydrated and then treated with microwave heating for retrieval of the antigens (Shi et al. 1991). The pre-treated slides were incubated overnight in the cold room with the IAP4 monoclonal antibodies in tissue culture supernatant diluted 1:2 with PBS. The IAP-anti-IAP complexes were then detected and visualized by means of biotin-streptavidin alkaline phosphatase anti-alkaline phosphatase staining, using a
Purification and characterization of the antibodies Immunoglobulins of IgM-type were partially purified as ascites as described by Neoh et al. (1986). Briefly, the antibodies in 10 ml ascites were precipitated at 0° C for 15 min using an equal volume of 25% polyethyleneglycol (PEG 6000) in VBS buffer [0.004 M 5,5-diethyl-barbituric acid buffer substance (Merck, Darmstadt, FRG), 0.15 M NaC1, 0.8 mM MgC12 and 0.3 mM CaC12, pH 7.2]; the protein pellet obtained after centrifugation was dissolved in VBS. The precipitation was repeated once and the resulting pellet was dissolved and stored in 10 ml 0.01 M phosphate-buffered saline (PBS) containing 0.5 M NaC1 and 20% glycerol. Isotypes were determined by ELISA technique with commercially available rabbit antibodies against mouse immunoglobulin subclasses (Bio-Rad Mouse Typer Kit, Richmond, Calif., USA). To assure immunochemical specificity, the reactivity of the antiIAP Mabs was tested by ELISA, using the pure antigens IAP, liver ALP and PLAP. The protein S-100, purified of bovine brain tissue, was included in the test as an unspecific protein.
• PLAP S-100