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Neuronal localization of CD38 antigen in the human brain
BRAIN RESEARCH Brain Research 697 (1995) 235-240
ELSEVIER
Research report
Neuronal localization of CD38 antigen in the human brain Masashi Mizuguchi a,b, *, Naruhito Otsuka b,c, Maroto Sato b Yoshifumi Ishii d, Shin-ichiro Kon d M i t s u n o r i Y a m a d a e, H i r o s h i N i s h i n a f, T o s h i a k i K a t a d a f, K a z u h i k o I k e d a b a Department of Mental Retardation and Birth Defect Research, National Institute ofNeuroscience, NCNP, Kodaira, Tokyo 187, Japan b Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, Tokyo, Japan c Department of Pathology, University of Tokyo, Tokyo, Japan d Department of Pathology, Sapporo Medical School, Sapporo,, Japan e Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan f Department of Life Science, Tokyo Institute of Technology, Tokyo, Japan Accepted 28 June 1995
Abstract
CD38 is a lymphocyte differentiation antigen which is involved in the cyclic ADP-ribose-mediated second messenger system. We provide immunochemical and immunohistochemical evidence for the expression of CD38 in the adult human brain. We used six polyclonal antibodies against synthetic CD38 polypeptides, in addition to four monoclonal antibodies already available. Brain CD38 was detectable by Western blotting after immunoaffinity purification of the brain extracts. Immunoperoxidase staining localized CD38 immunoreactivity to the perikarya and dendrites of many neurons, such as the cerebellar Purkinje cells, implying that CD38 is involved in the signal transduction within the central nervous system neurons. Keywords: Cyclic ADP-ribose; Second messenger system; Central nervous system; Immunohistochemistry; Western blotting; Neuron
1. Introduction
The human CD38 molecule is a 46 kDa type II singletransmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain [7,20]. This protein is an ectoenzyme with activities of ADP-ribosyl cyclase, cyclic ADP-ribose (cADPR) hydrolase and NAD glycohydrolase [5,9,18,21], and is involved in both the formation and hydrolysis of cADPR, a second messenger that regulates the mobilization of intracellular Ca 2+ [4]. Though originally identified as a T lymphocyte differentiation antigen [16], CD38 is expressed in a wide range of cells and tissues [11,17,19]. Its distribution in the nervous system, however, has not been established. Mammalian brains have potential receptors for cADPR, ryanodine receptors [14], but the enzyme responsible for cADPR metabolism in the brain has not been identified. CD38 is a candidate since an mRNA of its homologue is present in the rat brain [8,10]. In this study,
* Corresponding author. Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187, Japan. Fax: (81) (423) 46-1743. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
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we demonstrated the expression of CD38 antigen in adult human brain by immunochemical and immunohistochemical methods.
2. Materials and methods
2.1. Tissue samples Samples of human cerebral and cerebellar cortices were obtained at autopsy from four control patients, aged from 18 to 72 years. Samples of human thymus were removed at autopsy from a newborn infant at term.
2.2. Antibodies We prepared and characterized the 2D5 monoclonal antibody [6]. We purchased three commercially available monoclonals, Leul7 (clone HB-7), OKT10 and CD38 (clone T16), from Becton-Dickinson, Ortho Diagnostic Systems and Cosmo Bio, respectively. To obtain antibodies with a higher titer, we raised six polyclonal antibodies in rabbits against peptide fragments of CD38. The peptides
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were synthesized by solid-phase techniques according to the sequence deduced from human CD38 c D N A [7]; amino acid residues 1 - 1 5 ( M A N C E F S P V S G D K P C ; peptide KI) and 1 6 - 2 4 ( C R L S R R A Q L ; peptide N O / l ) , which are included in the cytoplasmic and transmembrane domains; residues 6 1 - 7 0 (with C-terminal cysteine, SGPGTT K R F P C ; peptide N O / 2 ) , 1 4 3 - 1 5 3 (with N-terminal cysteine, C F T L E D T L L G Y L ; peptide H R / 1 ) , 1 7 7 - 1 8 7 (with C-terminal cysteine, R K D C S N N P V S V C ; peptide M M ) and 2 8 7 - 3 0 0 ( C V K N P E D S S C T S E I ; peptide MS), which are included in the extracellular domain. They were coupled to keyhole limpet hemocyanin, and the animals received four subcutaneous injections of 1 mg conjugates. W e verified by enzyme-linked immunoassay that the antisera reacted specifically with corresponding peptides. Subsequently we examined by Western blotting whether they can recognize
glutathione S-transferase (GST)-CD38 (extracellular domain) fusion protein [9]. 2.3. W e s t e r n b l o t t i n g
Proteins were extracted with 100 m M Tris-HC1, pH 7.6, containing 1 m M EDTA, 1 m M phenylmethylsulfonyl fluoride and 1% Triton X-100, from the thymus and brain tissues, as well as CD38 + lymphoid cells such as Concanavalin A-stimulated peripheral blood lymphocytes, retinoic acid-stimulated U937 cells (myelomonocytic leukemia), Daudi cells (Burkitt's lymphoma) and K T - M immortalized B cells [13]. The proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane [13]. After blocking with 8% skim milk, the membrane was
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Fig. 1. Western blotting with anti-CD38 antibodies. (a) Blots of the lysates of E. coli transformed with either sense (lanes 1, 3, 5, 7 and 9) or anti-sense (lanes 2, 4, 6, 8 and 10) GST-CD38 expression vectors were incubated with T16 (lanes 1 and 2), MS (lanes 3 and 4), HR/1 (lanes 5 and 6), MM (lanes 7 and 8) and NO/2 (lanes 9 and 10) antibodies. Each of these antibodies detected the 54 kDa fusion protein produced by the sense vector. (b) Blots of the extracts of peripheral blood lymphocytes before (lane 1) or after (lane 2) stimulation with concanavalin A (10 p.g/ml), Daudi (lane 3), KT-M (lane 4) and U937 cells before (lane 5) and after (lane 6) stimulation with retinoic acid (0.5 /zM), and a human thymus homogenate (lane 7) were incubated with the NO/1 antibody. In all lanes, except lanes 1 and 5, CD38 antigen (46 kDa) was demonstrated. (c) Blots of the homogenates of thymus (lane 1), cerebrum (lane 2) and cerebellum (lane 4), and of the proteins immunoaffinity purified from cerebrum (lane 3) and cerebellum (lane 5) were incubated with the NO/1 antibody. A detectable amount of CD38 antigen was recovered in the immunoaffinity purified samples. The bands at 35-40 kDa may have resulted from proteolytic degradation or from co-purification of CD38-related molecules.
M. Mizuguchi et al. / Brain Research 697 (1995) 235-240
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incubated with one of the monoclonal (20 /xg/ml) or polyclonal (diluted 1:100) antibodies. The immunoproducts were visualized using an alkaline phosphatase-conjugated avidin-biotin complex and a bromochloroindolyl phosphate/nitroblue tetrazolium substrate (both from Vector).
alized with 0.03% diaminobenzidine-HC1 for 5-10 min. Control experiments were performed by substituting the first antibody with the preimmune or preabsorbed antisera.
2.4. Immunoaffinity purification
3.1. Western blotting and immunoaffinity purification
The homogenates of cerebral and cerebellar cortices were clarified by centrifugation at 105,000 × g for 30 min, and passed through an affinity column that consisted of the 2D5 monoclonal directly coupled to protein A beads (Pharmacia). After washing with the binding buffer, the bound proteins were eluted with 100 mM glycine buffer, pH 2.5, then collected in tubes containing 1 / 1 0 volume of 1 M phosphate buffer, pH 8.0.
Western blotting revealed that all the four antisera against the CD38 extracellular subsequences, N O / 2 , H R / 1 , MM and MS, as well as the T16 monoclonal, recognize the GST-CD38 fusion protein (Fig. la). The other monoclonals, HB-7 and OKT10, could not detect this antigen (data not shown). The N O / 1 antibody, which recognizes the junction between the cytoplasmic and transmembrane domains, was the only one that detected CD38 antigen in the lymphoid cells and tissues (Fig. lb). CD38 antigen in the brain was undetectable when homogenates of the cerebral and cerebellar tissues were blotted, but it was recovered after the homogenates were immunoaffinity purified using the 2D5 monoclonal (Fig. lc).
2.5. lmmunostaining
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 /xm-thick sections. Deparaffinized slides were immersed in 0.3% hydrogen peroxide in methanol for 20 min to inactivate endogenous peroxidase. After an incubation with 20% normal goat serum to block non-specific staining, the sections were successively incubated with one of the anti-CD38 monoclonal (diluted 1:10) or polyclonal (diluted 1:400-1,000), biotinylated horse anti-mouse IgG (Vector, diluted 1:200) or goat antirabbit IgG (Vector, diluted 1:500) and peroxidase-conjugated streptavidin (Zymed, diluted 1:500), each for 1 h at room temperature. The resultant immunoproduct ffas visu-
3. Results
3.2. Immunohistochemistry
Immunostaining of the thymus with all the monoclonal and polyclonal antibodies resulted in a similar profile, which was more intense in the cortex than in the medulla (Fig. 2a-c). In the cerebral and cerebellar cortex, CD38 immunoreactivity was positive in specific subpopulations of neurons. The T16 and 2D5 monoclonals, when incubated at high concentrations, weakly labeled the cytoplasm
Fig. 2. Immunostainingof human thymus with T16 (a), KI (b) and NO/2 (c) antibodies. Immunoreactivethymocyteswere more numerousin the cortex (top) than in the medulla (bottom). Hassall's corpuscleswere not stained. 92 ×
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Fig. 3. Immunostaining of human cerebellar cortex with T16 (a; 115 x ), NO/1 (b; 115 X ), N O / 2 (c; 184 X ), MM (d; 115 X ) and HR/1 (e; 369 X ). Purkinje and basket (arrows) cells were positively stained.
Fig. 4. Immunostaining of human cerebral cortex with T16 (a; 184 X ), NO/1 (b; 184 X ) and HR/1 (c; 369 X ) antibodies. Many neurons were stained.
M. Mizuguchi et al. / Brain Research 697 (1995) 235-240
of cerebellar Purkinje cells and cerebral cortical neurons (Figs. 3 and 4a). No positive staining was obtained with the HB-7 and OKT10. With all the polyclonal antibodies, the Purkinje cells were more intensely stained with a granular pattern. Positivity was also noted in the basket but not in the granular cells (Fig. 3 b - e ) . Immunoreactivity of the cerebral neurons was less intense, significant staining being obtained only with the N O / 1 and H R / 1 antibodies. Immunoreactive granules were abundant in the neuronal cytoplasm, and scattered throughout the neuropil (Fig. 4b,c). In negative control experiments, no positivity was observed in the slides incubated with the preimmune or preabsorbed antisera (data not shown).
4. Discussion Although CD38 antigen has been isolated from tissues other than the lymphoid system [21], the lack of antibodies that detect low levels of its expression on Western blots and on routine histological sections has hindered investigations into its in vivo distribution at the cellular and subcellular levels. Thus, most immunochemical investigations into CD38 have studied the antigen bound to the cell membrane of lymphoid cells, by surface-labeling cultured cells and by immunoprecipitaion [1,20]. However, accumulating evidence indicates that CD38 is transferred to localizations other than the cell membrane. CD38 undergoes internalization a n d / o r shedding, associated with the movements of other cell-surface molecules [3]. The presence of a soluble form of CD38 is also suspected [5,12]. Here we generated for the first time an anti-CD38 antibody, N O / l , that can specifically detect CD38 antigens on Western blots of tissue homogenates and cellular extracts. Using this antibody, we demonstrated the low but detectable level of CD38 expression in the cerebral and cerebellar tissues o f adult controls. Furthermore, the polyclonal antibodies ( N O / 1 and H R / 1 in particular) proved to be useful in immunostaining paraffin-embedded tissue sections. Our immunohistochemical investigations localized most o f the brain CD38 immunoreactivity into the perikaryal and dendritic cytoplasm o f neurons. The granular staining profile suggests an association with intracellular organelles. The neuronal localization of CD38 shown here is compatible with the notion that CD38 regulates the level of c A D P R within the perikarya and dendrites of neurons, which in turn mobilizes intracellular Ca 2+ and thereby controls brain functions, such as neuronal plasticity [2]. It is conceivable that alterations in the c A D P R - m e d i a t e d second messenger system occur in degenerating CNS neurons in certain pathologic conditions. In this context, it is notable that intense CD38 immunoreactivity is associated with neurofibrillary tangles, the pathologic hallmark of A l z h e i m e r ' s disease that occurs in the neuronal perikarya and proximal dendrites [15].
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Acknowledgements W e thank T. A i z a w a and S. Haga for technical assistance. This work was supported in part by the Grant-in-Aid (5670659) from the Ministry of Education, Science and Culture, Japan (to M.M.) and the Research Grant (6A-2) for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan (to K.I.).
References [1] Alessio, M., Roggero, S., Furano, A., De Monte, L.B., Peruzzi, L., Geuna, M., Malavasi, F., CD38 molecule: structural and biochemical analysis on human T lymphocytes, and plasma cells, J. Immunol., 145 (1990) 878-884. [2] Berridge, M.J., A tale of two messengers, Nature, 365 (1993) 388-389. [3] Furano, A., De Monte, L.B., Dianzani, U., Fomi, M. and Malavasi, F., Involvement of the multilineage CD38 molecule in a unique pathway of cell activation and proliferation, Eur. J. Immunol., 23 (1993) 2407-2411. [4] Galione, A., Cyclic ADP-ribose: a new way to control calcium, Science, 259 (1993) 325-326. [5] Howard, M., Grimaldi, J.C., Bazan, J.F., Lurid, F.E., SantosArgumedo, L., Parkhouse, R.M.E., Walseth, T.F. and Lee, H.C., Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38, Science, 262 (1993) 1056-1059. [6] Ishii, Y., Kon, S., Takei, T., Fujimoto, J. and Kikuchi, K., Four distinct antigen systems on human thymus and T cells defined by monoclonal antibodies: immunohistologieal and immunochemical studies, Clin. Exp. Immunol., 53 (1983) 31-40. [7] Jackson, D.G. and Bell, J.t., Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation~ J. Immunol., 144 (1990) 2811-2815. [8] Koguma, T., Takasawa, S., Tohgo, A., Karasawa, T., Furuya, Y., Yonekura, H. and Okamoto, H., Cloning and characterization of cDNA encoding rat ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (homologous to human CD38) from islets of Langerhans, Biochim. Biophys. Acta, 1223 (1994) 160-162. [9] Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K. and Katada, T., NAD glycohydrolase specifically induced by retinoic acid in human leukemic HL-60 cells, J. Biol. Chem., 268 (1993) 16895-16898. [10] Li, Q., Yamada, Y., Yasuda, K., Ihara, Y., Okamoto, Y., Kaisaki, P.J., Watanabe, R., Ikeda, K., Tsuda, K. and Seino, Y., A cloned rat CD38-homologous protein and its expression in pancreatic islets, Biochem. Biophys. Res. Commun., 202 (1994) 629-636. [11] Malavasi, F., Furano, A., Alessio, M., DeMonte, L.B., Ausiello, C.M., Dianzani, U., Lanza, F., Magrini, E., Momo, M. and Roggero, S., CD38: a multi-lineage cell activation molecule with a split personality, Int. J. Clin. Lab. Res., 22 (1992) 73-80. [12] Malavasi, F., Furano, A., Roggero, S., Horenstein, A., Calosso, L. and Mehta, K., Human CD38: a glycoprotein in search of a function, Immunol. Today, 15 (1994) 95-97. [13] Mizuguchi, M., Ikeda, K., Asada, M., Mizutani, S. and Kamoshita, S., Expression of Bcl-2 protein in murine neural cells in culture, Brain Res., 649 (1994) 197-202. [14] Nakanishi, S., Kuwajima, G. and Mikoshiba, K., Immunohistochemical localization of ryanodine receptors in mouse central nervous system, Neurosci. Res., 15 (1992) 130-142. [15] Otsuka, N., Mizuguchi, M., Aizawa, T., Haga, S., Sato, M., Inoya, H., Namba, Y., Machinami, R. and Ikeda, K., CD38 immunoreactivity in Alzheimer's neurofibrillary tangles (Abstr.), Brain Pathol., 4 (1994) 558.
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[16] Reinherz, E.L., Kung, P.C., Godstein, G., Levey, R.H. and Schlossman, S.F., Discrete stages of intrathymic differentiation: analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage, Proc. Natl. Acad. Sci. USA, 77 (1980) 1588-1592. [17] Stashenko, P., Nadler, L.M., Hardy, R. and Schlossman, S.F., Expression of cell surface markers after B lymphocyte differentiation, Proc. NatL Acad. Sci. USA, 78 (1981) 3848-3852. [18] Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H. and Okamoto, H., Synthesis and hydrolysis of cyclic ADP-ribose by human leukocyte antigen CD38 and inhibition of the hydrolysis by ATP, J. Biol. Chem., 268 (1993) 26052-26054.
[19] Teddler, T.F., Clement, L.T. and Cooper, M.D., Discontinuous expression of a membrane antigen (HB-7) during B lymphocyte differentiation, Tissue Antigens, 24 (1984) 140-149. [20] Terhorst, C., van Agthoven, A., LeClair, K., Snow, P., Reinherz, E. and Schlossman, S., Biochemical studies of the human thymocyte cell-surface antigens T6, T9 and T10, Cell, 23 (1981) 771-780. [21] Zocchi, E., Franco, L., Guida, L., Benatti, U., Bargellesi, A., Malavasi, F., Lee, H.C. and De Flora, A., A single protein immunologically identified as CD38 displays NAD + glycohydrolase, ADPribosyl cyclase and cyclic ADP-ribose hydrolase activities at the outer surface of human erythrocytes, Biochem. Biophys. Res. Commun., 196 (1993) 1459-1465.
ELSEVIER
Research report
Neuronal localization of CD38 antigen in the human brain Masashi Mizuguchi a,b, *, Naruhito Otsuka b,c, Maroto Sato b Yoshifumi Ishii d, Shin-ichiro Kon d M i t s u n o r i Y a m a d a e, H i r o s h i N i s h i n a f, T o s h i a k i K a t a d a f, K a z u h i k o I k e d a b a Department of Mental Retardation and Birth Defect Research, National Institute ofNeuroscience, NCNP, Kodaira, Tokyo 187, Japan b Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, Tokyo, Japan c Department of Pathology, University of Tokyo, Tokyo, Japan d Department of Pathology, Sapporo Medical School, Sapporo,, Japan e Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan f Department of Life Science, Tokyo Institute of Technology, Tokyo, Japan Accepted 28 June 1995
Abstract
CD38 is a lymphocyte differentiation antigen which is involved in the cyclic ADP-ribose-mediated second messenger system. We provide immunochemical and immunohistochemical evidence for the expression of CD38 in the adult human brain. We used six polyclonal antibodies against synthetic CD38 polypeptides, in addition to four monoclonal antibodies already available. Brain CD38 was detectable by Western blotting after immunoaffinity purification of the brain extracts. Immunoperoxidase staining localized CD38 immunoreactivity to the perikarya and dendrites of many neurons, such as the cerebellar Purkinje cells, implying that CD38 is involved in the signal transduction within the central nervous system neurons. Keywords: Cyclic ADP-ribose; Second messenger system; Central nervous system; Immunohistochemistry; Western blotting; Neuron
1. Introduction
The human CD38 molecule is a 46 kDa type II singletransmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain [7,20]. This protein is an ectoenzyme with activities of ADP-ribosyl cyclase, cyclic ADP-ribose (cADPR) hydrolase and NAD glycohydrolase [5,9,18,21], and is involved in both the formation and hydrolysis of cADPR, a second messenger that regulates the mobilization of intracellular Ca 2+ [4]. Though originally identified as a T lymphocyte differentiation antigen [16], CD38 is expressed in a wide range of cells and tissues [11,17,19]. Its distribution in the nervous system, however, has not been established. Mammalian brains have potential receptors for cADPR, ryanodine receptors [14], but the enzyme responsible for cADPR metabolism in the brain has not been identified. CD38 is a candidate since an mRNA of its homologue is present in the rat brain [8,10]. In this study,
* Corresponding author. Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187, Japan. Fax: (81) (423) 46-1743. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
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we demonstrated the expression of CD38 antigen in adult human brain by immunochemical and immunohistochemical methods.
2. Materials and methods
2.1. Tissue samples Samples of human cerebral and cerebellar cortices were obtained at autopsy from four control patients, aged from 18 to 72 years. Samples of human thymus were removed at autopsy from a newborn infant at term.
2.2. Antibodies We prepared and characterized the 2D5 monoclonal antibody [6]. We purchased three commercially available monoclonals, Leul7 (clone HB-7), OKT10 and CD38 (clone T16), from Becton-Dickinson, Ortho Diagnostic Systems and Cosmo Bio, respectively. To obtain antibodies with a higher titer, we raised six polyclonal antibodies in rabbits against peptide fragments of CD38. The peptides
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were synthesized by solid-phase techniques according to the sequence deduced from human CD38 c D N A [7]; amino acid residues 1 - 1 5 ( M A N C E F S P V S G D K P C ; peptide KI) and 1 6 - 2 4 ( C R L S R R A Q L ; peptide N O / l ) , which are included in the cytoplasmic and transmembrane domains; residues 6 1 - 7 0 (with C-terminal cysteine, SGPGTT K R F P C ; peptide N O / 2 ) , 1 4 3 - 1 5 3 (with N-terminal cysteine, C F T L E D T L L G Y L ; peptide H R / 1 ) , 1 7 7 - 1 8 7 (with C-terminal cysteine, R K D C S N N P V S V C ; peptide M M ) and 2 8 7 - 3 0 0 ( C V K N P E D S S C T S E I ; peptide MS), which are included in the extracellular domain. They were coupled to keyhole limpet hemocyanin, and the animals received four subcutaneous injections of 1 mg conjugates. W e verified by enzyme-linked immunoassay that the antisera reacted specifically with corresponding peptides. Subsequently we examined by Western blotting whether they can recognize
glutathione S-transferase (GST)-CD38 (extracellular domain) fusion protein [9]. 2.3. W e s t e r n b l o t t i n g
Proteins were extracted with 100 m M Tris-HC1, pH 7.6, containing 1 m M EDTA, 1 m M phenylmethylsulfonyl fluoride and 1% Triton X-100, from the thymus and brain tissues, as well as CD38 + lymphoid cells such as Concanavalin A-stimulated peripheral blood lymphocytes, retinoic acid-stimulated U937 cells (myelomonocytic leukemia), Daudi cells (Burkitt's lymphoma) and K T - M immortalized B cells [13]. The proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane [13]. After blocking with 8% skim milk, the membrane was
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Fig. 1. Western blotting with anti-CD38 antibodies. (a) Blots of the lysates of E. coli transformed with either sense (lanes 1, 3, 5, 7 and 9) or anti-sense (lanes 2, 4, 6, 8 and 10) GST-CD38 expression vectors were incubated with T16 (lanes 1 and 2), MS (lanes 3 and 4), HR/1 (lanes 5 and 6), MM (lanes 7 and 8) and NO/2 (lanes 9 and 10) antibodies. Each of these antibodies detected the 54 kDa fusion protein produced by the sense vector. (b) Blots of the extracts of peripheral blood lymphocytes before (lane 1) or after (lane 2) stimulation with concanavalin A (10 p.g/ml), Daudi (lane 3), KT-M (lane 4) and U937 cells before (lane 5) and after (lane 6) stimulation with retinoic acid (0.5 /zM), and a human thymus homogenate (lane 7) were incubated with the NO/1 antibody. In all lanes, except lanes 1 and 5, CD38 antigen (46 kDa) was demonstrated. (c) Blots of the homogenates of thymus (lane 1), cerebrum (lane 2) and cerebellum (lane 4), and of the proteins immunoaffinity purified from cerebrum (lane 3) and cerebellum (lane 5) were incubated with the NO/1 antibody. A detectable amount of CD38 antigen was recovered in the immunoaffinity purified samples. The bands at 35-40 kDa may have resulted from proteolytic degradation or from co-purification of CD38-related molecules.
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incubated with one of the monoclonal (20 /xg/ml) or polyclonal (diluted 1:100) antibodies. The immunoproducts were visualized using an alkaline phosphatase-conjugated avidin-biotin complex and a bromochloroindolyl phosphate/nitroblue tetrazolium substrate (both from Vector).
alized with 0.03% diaminobenzidine-HC1 for 5-10 min. Control experiments were performed by substituting the first antibody with the preimmune or preabsorbed antisera.
2.4. Immunoaffinity purification
3.1. Western blotting and immunoaffinity purification
The homogenates of cerebral and cerebellar cortices were clarified by centrifugation at 105,000 × g for 30 min, and passed through an affinity column that consisted of the 2D5 monoclonal directly coupled to protein A beads (Pharmacia). After washing with the binding buffer, the bound proteins were eluted with 100 mM glycine buffer, pH 2.5, then collected in tubes containing 1 / 1 0 volume of 1 M phosphate buffer, pH 8.0.
Western blotting revealed that all the four antisera against the CD38 extracellular subsequences, N O / 2 , H R / 1 , MM and MS, as well as the T16 monoclonal, recognize the GST-CD38 fusion protein (Fig. la). The other monoclonals, HB-7 and OKT10, could not detect this antigen (data not shown). The N O / 1 antibody, which recognizes the junction between the cytoplasmic and transmembrane domains, was the only one that detected CD38 antigen in the lymphoid cells and tissues (Fig. lb). CD38 antigen in the brain was undetectable when homogenates of the cerebral and cerebellar tissues were blotted, but it was recovered after the homogenates were immunoaffinity purified using the 2D5 monoclonal (Fig. lc).
2.5. lmmunostaining
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 /xm-thick sections. Deparaffinized slides were immersed in 0.3% hydrogen peroxide in methanol for 20 min to inactivate endogenous peroxidase. After an incubation with 20% normal goat serum to block non-specific staining, the sections were successively incubated with one of the anti-CD38 monoclonal (diluted 1:10) or polyclonal (diluted 1:400-1,000), biotinylated horse anti-mouse IgG (Vector, diluted 1:200) or goat antirabbit IgG (Vector, diluted 1:500) and peroxidase-conjugated streptavidin (Zymed, diluted 1:500), each for 1 h at room temperature. The resultant immunoproduct ffas visu-
3. Results
3.2. Immunohistochemistry
Immunostaining of the thymus with all the monoclonal and polyclonal antibodies resulted in a similar profile, which was more intense in the cortex than in the medulla (Fig. 2a-c). In the cerebral and cerebellar cortex, CD38 immunoreactivity was positive in specific subpopulations of neurons. The T16 and 2D5 monoclonals, when incubated at high concentrations, weakly labeled the cytoplasm
Fig. 2. Immunostainingof human thymus with T16 (a), KI (b) and NO/2 (c) antibodies. Immunoreactivethymocyteswere more numerousin the cortex (top) than in the medulla (bottom). Hassall's corpuscleswere not stained. 92 ×
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Fig. 3. Immunostaining of human cerebellar cortex with T16 (a; 115 x ), NO/1 (b; 115 X ), N O / 2 (c; 184 X ), MM (d; 115 X ) and HR/1 (e; 369 X ). Purkinje and basket (arrows) cells were positively stained.
Fig. 4. Immunostaining of human cerebral cortex with T16 (a; 184 X ), NO/1 (b; 184 X ) and HR/1 (c; 369 X ) antibodies. Many neurons were stained.
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of cerebellar Purkinje cells and cerebral cortical neurons (Figs. 3 and 4a). No positive staining was obtained with the HB-7 and OKT10. With all the polyclonal antibodies, the Purkinje cells were more intensely stained with a granular pattern. Positivity was also noted in the basket but not in the granular cells (Fig. 3 b - e ) . Immunoreactivity of the cerebral neurons was less intense, significant staining being obtained only with the N O / 1 and H R / 1 antibodies. Immunoreactive granules were abundant in the neuronal cytoplasm, and scattered throughout the neuropil (Fig. 4b,c). In negative control experiments, no positivity was observed in the slides incubated with the preimmune or preabsorbed antisera (data not shown).
4. Discussion Although CD38 antigen has been isolated from tissues other than the lymphoid system [21], the lack of antibodies that detect low levels of its expression on Western blots and on routine histological sections has hindered investigations into its in vivo distribution at the cellular and subcellular levels. Thus, most immunochemical investigations into CD38 have studied the antigen bound to the cell membrane of lymphoid cells, by surface-labeling cultured cells and by immunoprecipitaion [1,20]. However, accumulating evidence indicates that CD38 is transferred to localizations other than the cell membrane. CD38 undergoes internalization a n d / o r shedding, associated with the movements of other cell-surface molecules [3]. The presence of a soluble form of CD38 is also suspected [5,12]. Here we generated for the first time an anti-CD38 antibody, N O / l , that can specifically detect CD38 antigens on Western blots of tissue homogenates and cellular extracts. Using this antibody, we demonstrated the low but detectable level of CD38 expression in the cerebral and cerebellar tissues o f adult controls. Furthermore, the polyclonal antibodies ( N O / 1 and H R / 1 in particular) proved to be useful in immunostaining paraffin-embedded tissue sections. Our immunohistochemical investigations localized most o f the brain CD38 immunoreactivity into the perikaryal and dendritic cytoplasm o f neurons. The granular staining profile suggests an association with intracellular organelles. The neuronal localization of CD38 shown here is compatible with the notion that CD38 regulates the level of c A D P R within the perikarya and dendrites of neurons, which in turn mobilizes intracellular Ca 2+ and thereby controls brain functions, such as neuronal plasticity [2]. It is conceivable that alterations in the c A D P R - m e d i a t e d second messenger system occur in degenerating CNS neurons in certain pathologic conditions. In this context, it is notable that intense CD38 immunoreactivity is associated with neurofibrillary tangles, the pathologic hallmark of A l z h e i m e r ' s disease that occurs in the neuronal perikarya and proximal dendrites [15].
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Acknowledgements W e thank T. A i z a w a and S. Haga for technical assistance. This work was supported in part by the Grant-in-Aid (5670659) from the Ministry of Education, Science and Culture, Japan (to M.M.) and the Research Grant (6A-2) for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan (to K.I.).
References [1] Alessio, M., Roggero, S., Furano, A., De Monte, L.B., Peruzzi, L., Geuna, M., Malavasi, F., CD38 molecule: structural and biochemical analysis on human T lymphocytes, and plasma cells, J. Immunol., 145 (1990) 878-884. [2] Berridge, M.J., A tale of two messengers, Nature, 365 (1993) 388-389. [3] Furano, A., De Monte, L.B., Dianzani, U., Fomi, M. and Malavasi, F., Involvement of the multilineage CD38 molecule in a unique pathway of cell activation and proliferation, Eur. J. Immunol., 23 (1993) 2407-2411. [4] Galione, A., Cyclic ADP-ribose: a new way to control calcium, Science, 259 (1993) 325-326. [5] Howard, M., Grimaldi, J.C., Bazan, J.F., Lurid, F.E., SantosArgumedo, L., Parkhouse, R.M.E., Walseth, T.F. and Lee, H.C., Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38, Science, 262 (1993) 1056-1059. [6] Ishii, Y., Kon, S., Takei, T., Fujimoto, J. and Kikuchi, K., Four distinct antigen systems on human thymus and T cells defined by monoclonal antibodies: immunohistologieal and immunochemical studies, Clin. Exp. Immunol., 53 (1983) 31-40. [7] Jackson, D.G. and Bell, J.t., Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation~ J. Immunol., 144 (1990) 2811-2815. [8] Koguma, T., Takasawa, S., Tohgo, A., Karasawa, T., Furuya, Y., Yonekura, H. and Okamoto, H., Cloning and characterization of cDNA encoding rat ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (homologous to human CD38) from islets of Langerhans, Biochim. Biophys. Acta, 1223 (1994) 160-162. [9] Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K. and Katada, T., NAD glycohydrolase specifically induced by retinoic acid in human leukemic HL-60 cells, J. Biol. Chem., 268 (1993) 16895-16898. [10] Li, Q., Yamada, Y., Yasuda, K., Ihara, Y., Okamoto, Y., Kaisaki, P.J., Watanabe, R., Ikeda, K., Tsuda, K. and Seino, Y., A cloned rat CD38-homologous protein and its expression in pancreatic islets, Biochem. Biophys. Res. Commun., 202 (1994) 629-636. [11] Malavasi, F., Furano, A., Alessio, M., DeMonte, L.B., Ausiello, C.M., Dianzani, U., Lanza, F., Magrini, E., Momo, M. and Roggero, S., CD38: a multi-lineage cell activation molecule with a split personality, Int. J. Clin. Lab. Res., 22 (1992) 73-80. [12] Malavasi, F., Furano, A., Roggero, S., Horenstein, A., Calosso, L. and Mehta, K., Human CD38: a glycoprotein in search of a function, Immunol. Today, 15 (1994) 95-97. [13] Mizuguchi, M., Ikeda, K., Asada, M., Mizutani, S. and Kamoshita, S., Expression of Bcl-2 protein in murine neural cells in culture, Brain Res., 649 (1994) 197-202. [14] Nakanishi, S., Kuwajima, G. and Mikoshiba, K., Immunohistochemical localization of ryanodine receptors in mouse central nervous system, Neurosci. Res., 15 (1992) 130-142. [15] Otsuka, N., Mizuguchi, M., Aizawa, T., Haga, S., Sato, M., Inoya, H., Namba, Y., Machinami, R. and Ikeda, K., CD38 immunoreactivity in Alzheimer's neurofibrillary tangles (Abstr.), Brain Pathol., 4 (1994) 558.
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[16] Reinherz, E.L., Kung, P.C., Godstein, G., Levey, R.H. and Schlossman, S.F., Discrete stages of intrathymic differentiation: analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage, Proc. Natl. Acad. Sci. USA, 77 (1980) 1588-1592. [17] Stashenko, P., Nadler, L.M., Hardy, R. and Schlossman, S.F., Expression of cell surface markers after B lymphocyte differentiation, Proc. NatL Acad. Sci. USA, 78 (1981) 3848-3852. [18] Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H. and Okamoto, H., Synthesis and hydrolysis of cyclic ADP-ribose by human leukocyte antigen CD38 and inhibition of the hydrolysis by ATP, J. Biol. Chem., 268 (1993) 26052-26054.
[19] Teddler, T.F., Clement, L.T. and Cooper, M.D., Discontinuous expression of a membrane antigen (HB-7) during B lymphocyte differentiation, Tissue Antigens, 24 (1984) 140-149. [20] Terhorst, C., van Agthoven, A., LeClair, K., Snow, P., Reinherz, E. and Schlossman, S., Biochemical studies of the human thymocyte cell-surface antigens T6, T9 and T10, Cell, 23 (1981) 771-780. [21] Zocchi, E., Franco, L., Guida, L., Benatti, U., Bargellesi, A., Malavasi, F., Lee, H.C. and De Flora, A., A single protein immunologically identified as CD38 displays NAD + glycohydrolase, ADPribosyl cyclase and cyclic ADP-ribose hydrolase activities at the outer surface of human erythrocytes, Biochem. Biophys. Res. Commun., 196 (1993) 1459-1465.