Immunology 1990 69 391-395
Determination of the molecular nature and cellular localization of Thy-1 in human renal tissue T. MIYATA,*t K. ISOBE,* R. DAWSON, M. A. RITTER, R. INAGI,* 0. ODA,t R. TAGUCHI,§ H. IKEZAWA,§ I. INOUE,¶ H. SEO,T M. HASEGAWA,t S. KOBAYASHIt K. MAEDA,t K. YAMADAt & I. NAKASHIMA* Department of *Immunology, Nagoya University School of Medicine, Department of tInternal Medicine, Nagoya University School of Medicine, Branch Hospital, ¶Research Institute of Environmental Medicine, Nagoya University, tThe Bio-dynamic Research Institute, Nagoya Memorial Hospital, §Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan and Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K.
Acceptedfor publication 14 November 1989
SUMMARY The molecular nature and cellular localization of Thy-I antigen in human renal tissue were studied. Strong immunohistochemical staining was observed in frozen sections of human kidney using monoclonal anti-human Thy-1 antibody; this reaction was almost completely abolished by pretreating the kidney section with phosphatidyl inositol (PI)-specific phospholipase C (PI-PLC). Immunohistochemical analysis revealed that the Thy-I antigen is localized on the proximal tubular epithelial cells and the Bowman's capsule of the glomerulus. Northern blot analysis of renal mRNA using a cloned human Thy- I gene revealed the presence of human Thy- 1 mRNA of a similar size to the one in human brain. When a human kidney cDNA library was screened with the same probe, a cDNA of human Thy- I was isolated. Moreover, human Thy- I protein with a molecular weight (MW) of 21,000 was detected in renal tissue by gel electrophoresis and Western blot analysis using monoclonal anti-human Thy- I antibody. These data demonstrate for the first time the production of human Thy-I as a PI-anchored protein with a unique cellular location in human renal tissue.
INTRODUCTION
more, Thy- 1 antigen has been demonstrated in epithelium, muscle cells, fibroblasts and some connective tissue (Lennon, Unger & Dulbecco, 1978; Lesley & Lennon, 1977; Morris & Ritter, 1980; Ritter & Morris, 1980; Scheid et al., 1972; Stern, 1973; Walsh & Ritter, 1981). Marked inter-species variation has also been observed in renal tissue. No Thy- 1 has been demonstrated in renal tissue in the mouse (Dalchau & Fabre, 1979), but in rats Thy- 1 or Thy- 1like antigen has been demonstrated in the glomerular mesangial cells (MC) by immunofluorescence (Ishizaki et al., 1980; Morris & Ritter, 1980) and immunoelectron (Yamamoto et al., 1986) microscopy. In man, the existence of renal Thy-l-like antigen has been suggested by quantitative immunoabsorption analysis using anti-human brain sera (Dalchau & Fabre, 1979). However, no detailed analysis on its localization in renal tissue has been studied. The possibility that the serological demonstration of Thy-l specificity is due to cross-reaction at the determinant level has not been ruled out. In subsequent studies human Thy-I glycoprotein has been purified from human brain (Cotmore, Crowhurst & Waterfield, 1981), and the human genomic Thy-l gene has been cloned and sequenced (Rijs et al., 1985; Seki et al., 1985). More recently the Thy-1 molecule on rodent thymocytes has been shown to be phosphatidylinositol (PI)-anchored (Low & Kincade, 1985). However, the molecular nature and cellular localization of ThyI in human renal tissue remain to be defined.
The Thy-I molecule is a member of the immunoglobulin super family and may be closely related to the putative primordial immunoglobulin domain (Williams & Barclay, 1988). The molecule is present on a wide variety of tissues, and shows considerable inter-species variation in its distribution. However, despite detailed structural analysis, its exact function in these many locations is largely unknown. Thy-I is a major constituent of brain cell membranes in all species studied (Dalchau & Fabre, 1979; Morris, 1985). However, in lymphoid tissues its distribution shows considerable variation between species. Thy-I is the most abundant surface molecule of rodent thymocytes (Acton, Morris & Williams, 1974), and is found in reduced amounts on murine mature T lymphocytes (Raff, 1971), and rodent (Goldschneider, Gordon & Morris, 1978; Ritter, Morris & Goldschneider, 1980) and human (Foon et al., 1984; Ritter, Sauvage & Delia, 1983) early T and B lymphocytes and haematopoietic stem cells. FurtherAbbreviations: MC, mesangial cells; PI, phosphatidyl inositol; PIPLC, PI-specific phospholipase C; SDS, sodium dodecyl sulphate. Correspondence: Dr K. Isobe, Dept. of Immunology, Nagoya University, School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466, Japan.
391
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In this report, it will be presented that human renal tubular epithelium and glomerular Bowman's capsule express Thy- 1, as a PI-anchored protein, with structural properties similar to the one in the brain.
sis, the detection of Thy-I mRNA was carried out by Northern blot analysis (Maniatis et al., 1982) using the Bst ElI x Sac I fragment of cloned genomic human Thy-i gene (Rijs et al., 1985) as a probe.
MATERIALS AND METHODS
Construction of a human kidney cDNA library, isolation of a cDNA clone of human Thy-i and nucleotide sequence analysis Poly (A)+-mRNA was purified from total RNA of human kidney by oligo (dT) cellulose chromatography (Maniatis et al., 1982). cDNA was synthesized with randam hexanucleotides as a primer and ligated in bacteriophage gtlO under the conditions recommended by the suppliers (Amersham International plc, Amersham, Bucks, U.K.). The fragment of cloned genomic human Thy- I gene, described above, was also used to screen the library as a probe. Positively hybridized plaques were picked up and amplified with further purification (Maniatis et al., 1982). Insert cDNAs were subcloned into bacteriophage Ml3mpI8 and they were then sequenced by the dideoxy nucleotide strategy under the conditions recommended by the suppliers (TOYOBO,
Tissues
Adult human kidney was obtained by nephrectomy, dissected, snap-frozen and stored at - 700. This preparation showed normal kidney morphology under light microscopy. The renal tissue was examined for human Thy-I expression. Normal adult human brain and liver were also examined. Anti-human Thy-i monoclonal antibody BALB/c young female mice were immunized i.p. four times at approximately 4-week intervals with 107 DC-I cells. DC-I is a transformed mouse renal epithelial cell line that does not express the endogenous mouse Thy-1.2 molecule, but does express large quantities of human Thy-I due to the activation of a mouse/ human-coding 'ht' transgene (Kollias et al., 1987). Fusion of spleen cells with NOI myeloma cells, HAT selection and cloning were as described previously (De Maagd et al., 1985; Kohler & Milstein, 1975). Screening was by indirect immunoperoxidase analysis of frozen sections of human thymus. Specificity for human Thy-I was confirmed by Western blotting of NP-40 lysates from mouse DC-I cells and human thymus tissue. A large number of anti-human Thy-I cloned hybridomas was obtained, presumably due to the fact that Thy-I was the only human molecule on the immunizing cells. Clone B7 secretes a high-affinity IgG monoclonal anti-human Thy- I antibody, which was used throughout this study of human renal Thy-i.
PI-specific phospholipase C (PI-PLC) treatment PI-PLC was prepared from the culture supernatant of Bacillus thuringiensis as described elsewhere (Ikezawa et al., 1983). This preparation of PI-PLC gave a single band in polyacrylamide gel electrophoresis, and was protease-free (Ikezawa et al., 1983). Each kidney section was treated with 150 mU/ml af PI-PLC in phosphate-buffered saline (PBS), pH 7-2, at 370 for 1 hr. When mouse and rat thymocytes were treated with the PI-PLC, the Thy- I antigen, but not major histocompatibility complex (MHC) class I antigen, was released from their surface (data not shown). Immunohistochemical examination Renal tissue was fixed in periodate-lysine-8% paraformaldehyde (PLP) buffer for 6 hr at room temperature (McLean & Nakane, 1974), snap-frozen in liquid nitrogen and 3-p sections were cut by cryostat. The sections were stained with monoclonal anti-human Thy-1 antibody, biotinylated anti-mouse IgG and avidin-biotin peroxidase complex (ABC method) under the conditions recommended by the suppliers (Biogenecs Lab., Sanramon, CA). Northern blotting Total RNA from human kidney, brain and liver were prepared by the method of Maniatis, Fritsch & Sambrook (1982), with some modifications. Twenty micrograms of total RNA were subjected to 1 % agarose gel electrophoresis. After electrophore-
Osaka). Preparation of tissue proteins for electrophoresis The tissue (0-2 g) was homogenized and solubilized with 1-4 ml of 10 mm Tris-HCl buffer (pH 7 5) containing 2% 3-[(3cholamidopropyl) dimethylammonnio]- l-propanesulphonate (CHAPS; Sigma, St Louis, MO), 0-8 U micrococcal nuclease (Sigma), 0 05 mg RNase (Sigma) and 0 1 mg DNase (Sigma) for 5 min on ice. After centrifugation at 5000 g for 1 min at 40, the soluble material was lyophilized. The lyophilized sample was dissolved with 0 1 ml of 2% sodium dodesyl sulphate (SDS), 8 M urea solution. Insoluble substances were removed by centrifugation at 3000 g for 10 min. The protein concentration of the sample was estimated by the method of Bradford (1976). Fifty micrograms of tissue protein were applied to the SDS-polyacrylamide gel.
SDS-PAGE and immunoblot analysis SDS-PAGE was carried out by the method of Laemmli (1970) except for the conditions of gel concentration. A 4-17% polyacrylamide gradient gel containing 1% SDS and 8 M urea was used for separation of proteins. Three identical gels were electrophoresed at one time; one of the gels was stained with Coomassie brilliant blue (CBB) R-250 (Sigma). The proteins from the other gels were transferred to nitrocellulose membranes (Manabe, Takahashi & Okayama, 1984). The membrane was immunoblotted with monoclonal anti-human Thy-I antibody, biotinylated anti-mouse IgG and avidin-biotin-alkaline phosphatase complexes (ABC method) under the conditions recommended by the suppliers (Amersham International plc).
RESULTS
All kidney sections examined showed prominent Thy-I immunostaining, predominantly in the tubular epithelium and the Bowman's capsule of the glomerulus, when monoclonal antihuman Thy-i antibody (1:20 dilution) was used as the first antibody (Fig. lb, c). No staining was observed on control
Molecular characterization of Thy-i in human kidney
393
CY.j O.- .
-, s:
I 'i
Figure 1. Immunoperoxidase staining of human Thy-I in kidney sections. (a) Normal mouse serum (negative control); (b) and (c) monoclonal anti-human Thy- I antibody (no pretreatment with PI-PLC); (d) anti-human Thy- I antibody after pretreatment with PIPLC. The peroxidase reaction products were observed predominantly in the tubular epithelium cells and the Bowman's capsule of the glomerulus (b and c). In contrast, pretreatment with PI-PLC abolishes this reaction (d). Original magnification: (a), (b) and (d) x 200; (c) x 400.
28S-
2
1
Figure 2. Northern blot analysis of human brain, kidney and liver.
Ell
x
Sac I restriction
different tissues
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nylon membrane,
fragment
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electrophoresed
and
hybridized
in
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as
an
a
the
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agarose
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radiolabelled
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human liver RNA; lane 2, human brain RNA; lane 3, human RNA. The numbers indicate the
RNA.
position of
A Bst
Total RNA from to
a
Lane 1,
kidney
18 S and 28 S ribosomal
sections receiving normal mouse serum (1: 10) in place of monoclonal antibody (Fig. la). When kidney sections were pretreated with PI-PLC, definite Thy- I immunostaining was no longer observed (Fig. Id). The human Thy-I mRNA expression in the kidney by Northern blotting was analysed next (Fig. 2). The probe that contained the human Thy-i coding region hybridized with a single mRNA species of2kb in brain and kidney total RNA. The intensity of expression of human Thy-I mRNA in the latter was almost the same as that in the former. Thy-I mRNA was not detectable in the liver. Approximately 1 x t05 recombinants of human kidney cDNA library were screened. Three clones that hybridized strongly to genomic human Thy-I fragment were isolated. A cDNA insert with 205 bp was isolated from one clone, and its nucleotide sequence was determined. The sequence was completely identical to the second exon ofthe genomic human Thy- I gene reported previously (Seki et al., 1985) (Fig. 3). The molecular properties of human Thy-I in renal tissue were studied by Western blotting. The human Thy- I protein was identified by immunoblotting using monoclonal anti-human Thy-I antibody (Fig. 4b). Among the many bands from brain tissue that were detected on the gel by CBB staining (data not shown), two bands were labelled specifically with the antihuman Thy-I antibody (lane 2). The molecular weight of these bands were roughly estimated as 21,000 and 24,000, respectively. In renal tissue, only one Thy-I band was detected (lanes 3 and 4). The molecular weight of this band was estimated as 21,000, corresponding to the lower molecular weight protein
T. Miyata et al.
394
*-CTTGCAGGTC TCCCGAGGGC AGAAGGTGAC CAGCCTAACG GCCTGCCTAG TGGACCAGAG CCTTCGTCTG GACTGCCGCC ATGAGAATAC CAGCAGTTCA
CCCATCCAGT ACGAGTTCAG CCTGACCCGT GAGACAAAGA AGCACGTGCT CTTTGGCACT GTGGGGGTGC CTGAGCACAC ATACCGCTCC CGAACCAACT
TCACC-**
e
I
2
exon 2
exon 1
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exon
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Figure 3. Nucleotide sequence of the isolated clone of human Thy- I cDNA (*-**). Genomic human Thy- I et al., 1985).
(a)
(b)
MW
MW
94100068,00045,00030,000-
-24,000
-21,000
20,000 14,0001 2 3 4
1 2 34
Figure 4. SDS-PAGE and Western blot analysis of lysates of human brain, kidney and liver. Human Thy- I was detected by Western blotting with monoclonal anti-human Thy-I antibody (b). Normal mouse serum was used as control (a). Lane 1, human liver; lane 2, human brain; lanes 3 and 4, human kidney. The numbers indicate molecular weight of markers. The molecular weight ofhuman Thy- I proteins were estimated as 21,000 (brain and kidney) and 24,000 (brain), respectively.
detected in the brain. No band was observed in the liver (lane 1), or when anti-human Thy- I antibody was replaced with normal mouse serum (Fig. 4a). DISCUSSION This study defined the molecular nature and cellular localization of the Thy-I antigen in human renal tissue. Firstly, the human Thy-I antigen was demonstrated by immunohistochemistry to be localized in the proximal tubular epithelium and the epithelial cells of Bowman's capsule of the glomerulus. Secondly, this Thy- I antigen in human renal tissue was shown to be sensitive to PI-PLC, as previously demonstrated for Thy-I on rodent T lymphocytes (Low & Kincade, 1985). Thirdly, human
gene is shown
below (Seki
Thy-I mRNA, comparable to that of the brain, was present in renal tissue. The presence of such human Thy-I mRNA was confirmed by the isolation of a human Thy-I cDNA clone from a human kidney cDNA library. The nucleotide sequence of the isolated clone was completely identical to that of part of the second exon of genomic human Thy-I gene as reported previously (Seki et al., 1985). Lastly, human Thy-I protein was identified in extracts ofrenal tissue. The molecular weight of this renal Thy-I was 21,000, corresponding to the lower molecular weight band seen in human brain (this study) and comparable to previous data on human brain Thy-I (Cotmore et al., 1981). To our knowledge, this communication is therefore the first formal demonstration of human Thy-I in renal tissue. It has been reported that LAF-3, another PI-anchored protein, has an alternative, transmembranous form of structure (Dustin et al., 1987). However, the results presented here and those of Low & Kincade (1985) show that Thy- I on both human renal tissue and murine T lymphocytes is PI-anchored, and not anchored by a hydrophobic amino acid transmembrane domain. Interestingly, the molecular size of human Thy-I in renal tissue, determined by SDS-PAGE, was identical to the lower molecular form in the brain, where two forms were demonstrated. However, only a single species of mRNA was found in both tissues. The apparent difference between Thy-I in human kidney and brain must therefore be due to differential post-translational modification. Distinct glycosylation ofThy-I peptides from different tissues has been reported previously (Barclay, Letarte-Muirhead & Williams, 1976; Parekh et al., 1987). The function of Thy- I in renal tissue is a matter of speculation. Thy-i on rodent T lymphocytes is thought to play a role in the transduction of a cell activation/proliferation signal (Gunter et al., 1987), while it might also cause activation of the cell seeing it (Isobe et al., 1984). Alternatively, the presence of Thy- I on connective tissue has led to the suggestion that in these situations Thy-I may function as an adhesion molecule (Morris & Ritter, 1980; Ritter & Morris, 1980). ACKNOWLEDGMENT This work was supported in part by a Grant-in-aid from the Ministry of Education, Science and Culture.
Molecular characterization of Thy-i in human kidney REFERENCES ACTON R.T., MORRIS R.J. & WILLIAMS A.F. (1974) Estimation of the amount and tissue distribution of rat Thy- I antigen. Eur. J. Immunol. 4, 598. BARCLAY A.N., LETARTE-MUIRHEAD M. & WILLIAMS A.F. (1976) Chemical characterization of the Thy-1 glycoproteins from the membranes of rat thymocytes and brain. Nature (Lond.), 263, 563. BRADFORD M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248. COTMORE S.F., CROWHURST S.A. & WATERFIELD M.D. (1981) Purification of Thy-l-related glycoproteins from human brain and fibroblasts: comparisons between these molecules and murine glycoproteins carrying Thy- 1. 1 and Thy- 1.2 antigens. Eur. J. Immunol. 11,597. DALCHAU R. & FABRE J.W. (1979) Identification and unusual tissue distribution of the canine and human homologous of Thy-i . J. exp. Med. 147, 576. DE MAAGD R.A., MACKENZIE W.A., SCHUURMAN H.J., RITTER M.A., PRICE K.M., BROEKHUIZEN R. & KATER L. (1985) The human thymus microenvironment: heterogeneity detected by monoclonal anti-epithelial cell antibodies. Immunology, 54, 745. DUSTIN M.L., SELVARAJ P., MATTALIANO R.J. & SPRINGER T.A. (1987) Anchoring mechanisms for LFA-3 cell adhesion glycoprotein at membrane surface. Nature (Lond.), 329, 846. FOON K.A., NEUBAUER R.H., WLKSTRAND C.J., SCHROFF S.W., RABIN H. & SEEGER R.C. (1984) A monoclonal antibody recognizing human Thy- 1: distribution on human and non-human primate hematopoietic cells. J. Immunogen. 11, 233. GOLDSCHNEIDER I., GORDON L.K. & MORRIS R.J. (1978) Demonstration of Thy- I antigen on pluripotent haemopoietic stem cells in the rat. J. exp. Med. 148, 1351. GUNTER K.C., GERMAIN R.N., KROCZEK R.A., SAITO T., YOKOHAMA W.M., CHAN C., WEISS A. & SHEVACH E.M. (1987) Thy- I -mediated Tcell activation requires co-expression of CD/Ti complex. Nature (Lond.), 326, 505. IKEZAWA H., NAKABAYASHI T., SUZUKI K., NAKAJIMA M., TAGUCHI T. & TAGUCHI R. (1983) Complete purification of phosphatidyl-inositolspecific phospholipase C from a strain of Bacillus thuringiensis. J. Biochem. 93, 1717. ISHIZAKI M., SATO S., FUKUDA J., SUGISAKI Y. & MASUGI Y. (1980) The presence of Thy- 1. 1 antigen in rat glomerular mesangial cells. Biomed. Res. 1, 438. ISOBE K., NAKASHIMA I., NAGASE F., KATO N., MIZOGUCHI K., KAWASHIMA K. & LAKE P. (1984) Cellular mechanism of primary anti-Thy- I antibody responses in vitro induced by uniquely immunogenic thymocyte antigens. J. Immunol. 132, 1 100. KOHLER G. & MILSTEIN C. (1975) Continuous culture of fused cells secreting antibody of predefined specificity. Nature (Lond.), 256, 497. KOLLIAS G., SPANOPOULOU E., GROSVELD F., RITTER M., BEECH J. & MORRIS R. (1987) Differential regulation of a Thy- I gene in transgenic mice. Proc. natl. Acad. Sci. U.S.A. 84, 1492. LAEMMLI U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophase T4. Nature (Lond.), 227, 680. LENNON V.A., UNGER M. & DULBECCO R. (1978) Thy-i: a differentiation marker of potential mammary myoepithelial cells in vitro. Proc. natl. Acad. Sci. U.S.A. 75, 6093.
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LESLEY J.F. & LENNON V.A. (1977) Transitory expression of Thy-1 antigen in skeletal muscle development. Nature (Lond.), 268, 163. Low M.G. & KINCADE P.W. (1985) Phosphatidylinositol is the membrane-anchoring domain of the Thy-i glycoprotein. Nature (Lond.), 318, 62. MCLEAN I.W. & NAKANE P.K. (1974) Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J. Histchem. Cytochem. 22, 1077. MANABE T., TAKAHASHI Y. & OKAYAMA T. (1984) An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gel. Anal. Biochem. 143,39. MANIATIS T., FRITSCH E.F. & SAMBROOK J. (1982) Extraction, purification, and analysis of mRNA from eukaryotic cells. In: Molecular Cloning, pp. 188-209. Cold Spring Harbor Laboratory, NY. MORRIS R. (1985) Thy-i in developing nervous tissue. Dev. Neurosci. 7, 133. MORRIS R. & RITTER M. (1980) Association of Thy-i cell surface differentiation antigen with certain connective tissues in vivo. Cell Tissue Res. 206, 459. PAREKH R.B., TSE A.G.D., DWEK R.A., WILLIAMS A.F. & RADEMACHER T.W. (1987) Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy- 1. EMBO. J. 6, 1233. RAFF M.C. (1971) Surface antigen markers for distinguishing T and B lymphocytes in mice. Transplant. Rev. 6, 53. RIJs J.V., GIGUERE V., HURST J., AGTHOVEN T.V., KESSEL A.D.V., GOYERT S. & GROSVELD F. (1985) Chromosomal localization of the human Thy-i gene. Proc. natl. Acad. Sci. U.S.A. 82, 5832. RITTER M.A. & MORRIS R.J. (1980) Thy-I antigen: selective association in lymphoid organs with the vascular basement membrane involved in lymphocyte recirculation. Immunology, 39, 85. RITTER M.A., MORRIS R.J. & GOLDSCHNEIDER I. (1980) Hidden Thy-I antigen is a subpopulation of mouse bone marrow cells. Immunology, 39, 375. RITTER M.A., SAUVAGE C.A. & DELIA D. (1983) Human Thy-I antigen: cell surface expression on early T and B lymphocytes. Immunology, 49, 555. SCHEID M., BOYSE E.A., CARSWELL E.A. & OLD L.J. (1972) Serologically demonstrable alloantigens of mouse epidermal cells. J. exp. Med. 135, 938. SEKI T., SPURR N., OBATA F., GOYERT S., GOODFELLOW P. & SILVER J. (1985) The human Thy-i gene: structure and chromosomal location Proc. natl. Acad. Sci. U.S.A. 82, 6657. STERN P.L. (1973) Thy-l alloantigen on mouse and rat fibroblasts. Nature New Biol. 246, 76. WALSH F.S. & RITTER M.A. (1981) Surface antigen differentiation during human myogenesis in culture. Nature (Lond.), 289, 60. WILLIAMS A.F. & BARCLAY A.N. (1988) The immunoglobulin superfamily-domain for cell surface recognition. Ann. Rev. Immunol. 6, 381. YAMAMOTO T., YAMAMOTO K., KAWASAKI K., YAOITA E., SHIMIzu F. & KIHARA I. (1986) Immunoelectron microscopic demonstration of Thy- I antigen on the surfaces of mesangial cells in the rat glomeruli. Nephron, 43, 293.
Determination of the molecular nature and cellular localization of Thy-1 in human renal tissue T. MIYATA,*t K. ISOBE,* R. DAWSON, M. A. RITTER, R. INAGI,* 0. ODA,t R. TAGUCHI,§ H. IKEZAWA,§ I. INOUE,¶ H. SEO,T M. HASEGAWA,t S. KOBAYASHIt K. MAEDA,t K. YAMADAt & I. NAKASHIMA* Department of *Immunology, Nagoya University School of Medicine, Department of tInternal Medicine, Nagoya University School of Medicine, Branch Hospital, ¶Research Institute of Environmental Medicine, Nagoya University, tThe Bio-dynamic Research Institute, Nagoya Memorial Hospital, §Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan and Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K.
Acceptedfor publication 14 November 1989
SUMMARY The molecular nature and cellular localization of Thy-I antigen in human renal tissue were studied. Strong immunohistochemical staining was observed in frozen sections of human kidney using monoclonal anti-human Thy-1 antibody; this reaction was almost completely abolished by pretreating the kidney section with phosphatidyl inositol (PI)-specific phospholipase C (PI-PLC). Immunohistochemical analysis revealed that the Thy-I antigen is localized on the proximal tubular epithelial cells and the Bowman's capsule of the glomerulus. Northern blot analysis of renal mRNA using a cloned human Thy- I gene revealed the presence of human Thy- 1 mRNA of a similar size to the one in human brain. When a human kidney cDNA library was screened with the same probe, a cDNA of human Thy- I was isolated. Moreover, human Thy- I protein with a molecular weight (MW) of 21,000 was detected in renal tissue by gel electrophoresis and Western blot analysis using monoclonal anti-human Thy- I antibody. These data demonstrate for the first time the production of human Thy-I as a PI-anchored protein with a unique cellular location in human renal tissue.
INTRODUCTION
more, Thy- 1 antigen has been demonstrated in epithelium, muscle cells, fibroblasts and some connective tissue (Lennon, Unger & Dulbecco, 1978; Lesley & Lennon, 1977; Morris & Ritter, 1980; Ritter & Morris, 1980; Scheid et al., 1972; Stern, 1973; Walsh & Ritter, 1981). Marked inter-species variation has also been observed in renal tissue. No Thy- 1 has been demonstrated in renal tissue in the mouse (Dalchau & Fabre, 1979), but in rats Thy- 1 or Thy- 1like antigen has been demonstrated in the glomerular mesangial cells (MC) by immunofluorescence (Ishizaki et al., 1980; Morris & Ritter, 1980) and immunoelectron (Yamamoto et al., 1986) microscopy. In man, the existence of renal Thy-l-like antigen has been suggested by quantitative immunoabsorption analysis using anti-human brain sera (Dalchau & Fabre, 1979). However, no detailed analysis on its localization in renal tissue has been studied. The possibility that the serological demonstration of Thy-l specificity is due to cross-reaction at the determinant level has not been ruled out. In subsequent studies human Thy-I glycoprotein has been purified from human brain (Cotmore, Crowhurst & Waterfield, 1981), and the human genomic Thy-l gene has been cloned and sequenced (Rijs et al., 1985; Seki et al., 1985). More recently the Thy-1 molecule on rodent thymocytes has been shown to be phosphatidylinositol (PI)-anchored (Low & Kincade, 1985). However, the molecular nature and cellular localization of ThyI in human renal tissue remain to be defined.
The Thy-I molecule is a member of the immunoglobulin super family and may be closely related to the putative primordial immunoglobulin domain (Williams & Barclay, 1988). The molecule is present on a wide variety of tissues, and shows considerable inter-species variation in its distribution. However, despite detailed structural analysis, its exact function in these many locations is largely unknown. Thy-I is a major constituent of brain cell membranes in all species studied (Dalchau & Fabre, 1979; Morris, 1985). However, in lymphoid tissues its distribution shows considerable variation between species. Thy-I is the most abundant surface molecule of rodent thymocytes (Acton, Morris & Williams, 1974), and is found in reduced amounts on murine mature T lymphocytes (Raff, 1971), and rodent (Goldschneider, Gordon & Morris, 1978; Ritter, Morris & Goldschneider, 1980) and human (Foon et al., 1984; Ritter, Sauvage & Delia, 1983) early T and B lymphocytes and haematopoietic stem cells. FurtherAbbreviations: MC, mesangial cells; PI, phosphatidyl inositol; PIPLC, PI-specific phospholipase C; SDS, sodium dodecyl sulphate. Correspondence: Dr K. Isobe, Dept. of Immunology, Nagoya University, School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466, Japan.
391
392
T. Miyata et al.
In this report, it will be presented that human renal tubular epithelium and glomerular Bowman's capsule express Thy- 1, as a PI-anchored protein, with structural properties similar to the one in the brain.
sis, the detection of Thy-I mRNA was carried out by Northern blot analysis (Maniatis et al., 1982) using the Bst ElI x Sac I fragment of cloned genomic human Thy-i gene (Rijs et al., 1985) as a probe.
MATERIALS AND METHODS
Construction of a human kidney cDNA library, isolation of a cDNA clone of human Thy-i and nucleotide sequence analysis Poly (A)+-mRNA was purified from total RNA of human kidney by oligo (dT) cellulose chromatography (Maniatis et al., 1982). cDNA was synthesized with randam hexanucleotides as a primer and ligated in bacteriophage gtlO under the conditions recommended by the suppliers (Amersham International plc, Amersham, Bucks, U.K.). The fragment of cloned genomic human Thy- I gene, described above, was also used to screen the library as a probe. Positively hybridized plaques were picked up and amplified with further purification (Maniatis et al., 1982). Insert cDNAs were subcloned into bacteriophage Ml3mpI8 and they were then sequenced by the dideoxy nucleotide strategy under the conditions recommended by the suppliers (TOYOBO,
Tissues
Adult human kidney was obtained by nephrectomy, dissected, snap-frozen and stored at - 700. This preparation showed normal kidney morphology under light microscopy. The renal tissue was examined for human Thy-I expression. Normal adult human brain and liver were also examined. Anti-human Thy-i monoclonal antibody BALB/c young female mice were immunized i.p. four times at approximately 4-week intervals with 107 DC-I cells. DC-I is a transformed mouse renal epithelial cell line that does not express the endogenous mouse Thy-1.2 molecule, but does express large quantities of human Thy-I due to the activation of a mouse/ human-coding 'ht' transgene (Kollias et al., 1987). Fusion of spleen cells with NOI myeloma cells, HAT selection and cloning were as described previously (De Maagd et al., 1985; Kohler & Milstein, 1975). Screening was by indirect immunoperoxidase analysis of frozen sections of human thymus. Specificity for human Thy-I was confirmed by Western blotting of NP-40 lysates from mouse DC-I cells and human thymus tissue. A large number of anti-human Thy-I cloned hybridomas was obtained, presumably due to the fact that Thy-I was the only human molecule on the immunizing cells. Clone B7 secretes a high-affinity IgG monoclonal anti-human Thy- I antibody, which was used throughout this study of human renal Thy-i.
PI-specific phospholipase C (PI-PLC) treatment PI-PLC was prepared from the culture supernatant of Bacillus thuringiensis as described elsewhere (Ikezawa et al., 1983). This preparation of PI-PLC gave a single band in polyacrylamide gel electrophoresis, and was protease-free (Ikezawa et al., 1983). Each kidney section was treated with 150 mU/ml af PI-PLC in phosphate-buffered saline (PBS), pH 7-2, at 370 for 1 hr. When mouse and rat thymocytes were treated with the PI-PLC, the Thy- I antigen, but not major histocompatibility complex (MHC) class I antigen, was released from their surface (data not shown). Immunohistochemical examination Renal tissue was fixed in periodate-lysine-8% paraformaldehyde (PLP) buffer for 6 hr at room temperature (McLean & Nakane, 1974), snap-frozen in liquid nitrogen and 3-p sections were cut by cryostat. The sections were stained with monoclonal anti-human Thy-1 antibody, biotinylated anti-mouse IgG and avidin-biotin peroxidase complex (ABC method) under the conditions recommended by the suppliers (Biogenecs Lab., Sanramon, CA). Northern blotting Total RNA from human kidney, brain and liver were prepared by the method of Maniatis, Fritsch & Sambrook (1982), with some modifications. Twenty micrograms of total RNA were subjected to 1 % agarose gel electrophoresis. After electrophore-
Osaka). Preparation of tissue proteins for electrophoresis The tissue (0-2 g) was homogenized and solubilized with 1-4 ml of 10 mm Tris-HCl buffer (pH 7 5) containing 2% 3-[(3cholamidopropyl) dimethylammonnio]- l-propanesulphonate (CHAPS; Sigma, St Louis, MO), 0-8 U micrococcal nuclease (Sigma), 0 05 mg RNase (Sigma) and 0 1 mg DNase (Sigma) for 5 min on ice. After centrifugation at 5000 g for 1 min at 40, the soluble material was lyophilized. The lyophilized sample was dissolved with 0 1 ml of 2% sodium dodesyl sulphate (SDS), 8 M urea solution. Insoluble substances were removed by centrifugation at 3000 g for 10 min. The protein concentration of the sample was estimated by the method of Bradford (1976). Fifty micrograms of tissue protein were applied to the SDS-polyacrylamide gel.
SDS-PAGE and immunoblot analysis SDS-PAGE was carried out by the method of Laemmli (1970) except for the conditions of gel concentration. A 4-17% polyacrylamide gradient gel containing 1% SDS and 8 M urea was used for separation of proteins. Three identical gels were electrophoresed at one time; one of the gels was stained with Coomassie brilliant blue (CBB) R-250 (Sigma). The proteins from the other gels were transferred to nitrocellulose membranes (Manabe, Takahashi & Okayama, 1984). The membrane was immunoblotted with monoclonal anti-human Thy-I antibody, biotinylated anti-mouse IgG and avidin-biotin-alkaline phosphatase complexes (ABC method) under the conditions recommended by the suppliers (Amersham International plc).
RESULTS
All kidney sections examined showed prominent Thy-I immunostaining, predominantly in the tubular epithelium and the Bowman's capsule of the glomerulus, when monoclonal antihuman Thy-i antibody (1:20 dilution) was used as the first antibody (Fig. lb, c). No staining was observed on control
Molecular characterization of Thy-i in human kidney
393
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Figure 1. Immunoperoxidase staining of human Thy-I in kidney sections. (a) Normal mouse serum (negative control); (b) and (c) monoclonal anti-human Thy- I antibody (no pretreatment with PI-PLC); (d) anti-human Thy- I antibody after pretreatment with PIPLC. The peroxidase reaction products were observed predominantly in the tubular epithelium cells and the Bowman's capsule of the glomerulus (b and c). In contrast, pretreatment with PI-PLC abolishes this reaction (d). Original magnification: (a), (b) and (d) x 200; (c) x 400.
28S-
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Figure 2. Northern blot analysis of human brain, kidney and liver.
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sections receiving normal mouse serum (1: 10) in place of monoclonal antibody (Fig. la). When kidney sections were pretreated with PI-PLC, definite Thy- I immunostaining was no longer observed (Fig. Id). The human Thy-I mRNA expression in the kidney by Northern blotting was analysed next (Fig. 2). The probe that contained the human Thy-i coding region hybridized with a single mRNA species of2kb in brain and kidney total RNA. The intensity of expression of human Thy-I mRNA in the latter was almost the same as that in the former. Thy-I mRNA was not detectable in the liver. Approximately 1 x t05 recombinants of human kidney cDNA library were screened. Three clones that hybridized strongly to genomic human Thy-I fragment were isolated. A cDNA insert with 205 bp was isolated from one clone, and its nucleotide sequence was determined. The sequence was completely identical to the second exon ofthe genomic human Thy- I gene reported previously (Seki et al., 1985) (Fig. 3). The molecular properties of human Thy-I in renal tissue were studied by Western blotting. The human Thy- I protein was identified by immunoblotting using monoclonal anti-human Thy-I antibody (Fig. 4b). Among the many bands from brain tissue that were detected on the gel by CBB staining (data not shown), two bands were labelled specifically with the antihuman Thy-I antibody (lane 2). The molecular weight of these bands were roughly estimated as 21,000 and 24,000, respectively. In renal tissue, only one Thy-I band was detected (lanes 3 and 4). The molecular weight of this band was estimated as 21,000, corresponding to the lower molecular weight protein
T. Miyata et al.
394
*-CTTGCAGGTC TCCCGAGGGC AGAAGGTGAC CAGCCTAACG GCCTGCCTAG TGGACCAGAG CCTTCGTCTG GACTGCCGCC ATGAGAATAC CAGCAGTTCA
CCCATCCAGT ACGAGTTCAG CCTGACCCGT GAGACAAAGA AGCACGTGCT CTTTGGCACT GTGGGGGTGC CTGAGCACAC ATACCGCTCC CGAACCAACT
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Figure 3. Nucleotide sequence of the isolated clone of human Thy- I cDNA (*-**). Genomic human Thy- I et al., 1985).
(a)
(b)
MW
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-24,000
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Figure 4. SDS-PAGE and Western blot analysis of lysates of human brain, kidney and liver. Human Thy- I was detected by Western blotting with monoclonal anti-human Thy-I antibody (b). Normal mouse serum was used as control (a). Lane 1, human liver; lane 2, human brain; lanes 3 and 4, human kidney. The numbers indicate molecular weight of markers. The molecular weight ofhuman Thy- I proteins were estimated as 21,000 (brain and kidney) and 24,000 (brain), respectively.
detected in the brain. No band was observed in the liver (lane 1), or when anti-human Thy- I antibody was replaced with normal mouse serum (Fig. 4a). DISCUSSION This study defined the molecular nature and cellular localization of the Thy-I antigen in human renal tissue. Firstly, the human Thy-I antigen was demonstrated by immunohistochemistry to be localized in the proximal tubular epithelium and the epithelial cells of Bowman's capsule of the glomerulus. Secondly, this Thy- I antigen in human renal tissue was shown to be sensitive to PI-PLC, as previously demonstrated for Thy-I on rodent T lymphocytes (Low & Kincade, 1985). Thirdly, human
gene is shown
below (Seki
Thy-I mRNA, comparable to that of the brain, was present in renal tissue. The presence of such human Thy-I mRNA was confirmed by the isolation of a human Thy-I cDNA clone from a human kidney cDNA library. The nucleotide sequence of the isolated clone was completely identical to that of part of the second exon of genomic human Thy-I gene as reported previously (Seki et al., 1985). Lastly, human Thy-I protein was identified in extracts ofrenal tissue. The molecular weight of this renal Thy-I was 21,000, corresponding to the lower molecular weight band seen in human brain (this study) and comparable to previous data on human brain Thy-I (Cotmore et al., 1981). To our knowledge, this communication is therefore the first formal demonstration of human Thy-I in renal tissue. It has been reported that LAF-3, another PI-anchored protein, has an alternative, transmembranous form of structure (Dustin et al., 1987). However, the results presented here and those of Low & Kincade (1985) show that Thy- I on both human renal tissue and murine T lymphocytes is PI-anchored, and not anchored by a hydrophobic amino acid transmembrane domain. Interestingly, the molecular size of human Thy-I in renal tissue, determined by SDS-PAGE, was identical to the lower molecular form in the brain, where two forms were demonstrated. However, only a single species of mRNA was found in both tissues. The apparent difference between Thy-I in human kidney and brain must therefore be due to differential post-translational modification. Distinct glycosylation ofThy-I peptides from different tissues has been reported previously (Barclay, Letarte-Muirhead & Williams, 1976; Parekh et al., 1987). The function of Thy- I in renal tissue is a matter of speculation. Thy-i on rodent T lymphocytes is thought to play a role in the transduction of a cell activation/proliferation signal (Gunter et al., 1987), while it might also cause activation of the cell seeing it (Isobe et al., 1984). Alternatively, the presence of Thy- I on connective tissue has led to the suggestion that in these situations Thy-I may function as an adhesion molecule (Morris & Ritter, 1980; Ritter & Morris, 1980). ACKNOWLEDGMENT This work was supported in part by a Grant-in-aid from the Ministry of Education, Science and Culture.
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