BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
246, 501–506 (1998)
RC988648
Expression of Extracellular Calcium (Ca2/ o )-Sensing Receptor in Human Peripheral Blood Monocytes Toru Yamaguchi,*,1 Ivona Olozak,† Naibedya Chattopadhyay,* Robert R. Butters,* Olga Kifor,* David T. Scadden,† and Edward M. Brown* *Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, and †Experimental Hematology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02115
Received April 3, 1998
The calcium-sensing receptor (CaR) is a G proteincoupled receptor playing key roles in extracellular calcium ion (Ca2/ o ) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the ‘‘reversal’’ phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2/ o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2/ o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2/ o -sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2/ o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also ex1 To whom correspondence should be addressed. Fax: 1-617-7325764. E-mail:
[email protected].
press the CaR, the latter might play some role in the ‘‘reversal’’ phase of bone remodeling, sensing local changes in Ca2/ resulting from osteoclastic bone reo sorption and secreting osteotropic cytokines or performing other Ca2/ o -regulated functions that contribute to the control of bone turnover. q 1998 Academic Press
The purpose of the present study was to reevaluate the expression of the extracellular calcium (Cao2/)-sensing receptor in human peripheral blood monocytes. The CaR is a G protein-coupled receptor playing key roles in Cao2/ homeostasis in parathyroid gland and kidney (1). Bone, like parathyroid and kidney, is involved in systemic mineral ion homeostasis (2), and thus it is possible that the CaR might also play some role within the skeleton by sensing local changes in Cao2/ caused by bone remodeling. We recently reported that the CaR is expressed in nonspecific-esterase positive, macrophage-like mononuclear cells that were cultured from murine bone marrow (3). Such cells could be involved in mineral ion or skeletal homeostasis by serving as osteoclast precursors, which are known to arise from cells of this same lineage (4), and/or by secreting cytokines, such as interleukin-1 (IL-1) or interleukin-6 (IL6) (5), that have important actions on bone cells. Of all the cell types present in bone marrow, macrophagelike mononuclear cells are particularly good candidates for sensing local changes in Cao2/, because they appear at sites of osteoclastic bone resorption during the ‘‘reversal’’ phase of bone remodeling following osteoclastic bone resorption (6), which can produce local increases in Cao2/ within the immediate vicinity of osteoclasts reaching levels as high as 40 mM (7). Peripheral blood monocytes are thought to provide a valuable model of similar cells that are present in bone marrow and play key roles in regulating bone turnover. For example, Pacifici et al. have shown that that IL-1 activity released from human peripheral blood mono-
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cytes reflected bone formation rate in osteoporotic patients (8). In addition, bone matrix constituents stimulated IL-1 release from human blood mononuclear cells (9). Finally, Fujikawa, et al. showed that human peripheral blood monocytes retained the same character as bone marrow-derived cells of the macrophage/monocyte lineage, because a subset of peripheral monocytes had the capacity to differentiate into mature functional osteoclasts under specific culture conditions (10). Previous studies have described several effects of Cao2/ on peripheral blood monocytes. Sugimoto et al. showed that high Cao2/ induced the chemotaxis of human peripheral blood monocytes (11) and that conditioned medium from monocytes cultured at high Cao2/ (which contains humoral factors such as ILs) stimulated DNA synthesis and alkaline phosphatase activity in osteoblastic MC3T3-E1 cells (11, 12) and inhibited the formation of osteoclast-like cells from their precursors (13). Bornefalk, et al. have recently shown that raising Cao2/, either in vivo or in vitro, stimulated the release of interleukin-6 (IL-6) from peripheral blood monocytes (14), providing further evidence that these cells express a Cao2/-sensing mechanism that could mediate direct actions of Cao2/ on monocyte function relevant to skeletal turnover. In the latter study, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in monocytes (14). Since we recently demonstrated that monocytes cultured from murine marrow expressed the CaR (3), in the present study we have utilized immunocytochemistry, flow cytometry, and Western analysis with a CaRspecific antiserum as well as reverse-transcriptionpolymerase chain reaction (RT-PCR) with CaR-specific primers to document the presence of CaR protein and transcripts, respectively, in monocytes isolated from human peripheral blood. MATERIALS AND METHODS Human peripheral blood monocyte culture. Human peripheral blood monocytes derived from healthy men were cultured as previously described in detail (15). In brief, freshly drawn heparinized (10 U/ml) blood was fractionated on Histopaque 1077 (Sigma Chemical Co., St. Louis, MO), and the low density peripheral blood mononuclear cells were removed from the interface, according to the manufacturer’s instructions. The cells were resuspended in RPMI 1640 medium (GIBCO/BRL, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT) and 1% penicillin/streptomycin (GIBCO/ BRL) at a concentration of 2 1 104 cells/ml. Seven ml of the cell suspension was seeded into a 25 cm2 culture flask in 5% CO2 at 377C for Western blot or RT-PCR analysis. For morphological evaluation, 500 ml of the cell suspension was plated onto a 12 mm circular glass cover slip in a 24-well (2.0 cm2) plate. After 24 hours of culture, the medium containing non-adherent cells was discarded, and adherent cells were subjected to further experimentation as described below. Non-specific esterase (NSE) staining of the adherent cells showed that they consisted of Ç90% monocytes (viz., Fig. 1C). Immunocytochemistry for CaR in human peripheral blood monocytes. A CaR-specific polyclonal antiserum (4637) was generously
provided by Drs. Forrest Fuller and Karen Krapcho of NPS Pharmaceuticals, Inc., Salt Lake City, UT. This antiserum was raised against a peptide (FF-7) corresponding to amino acids 345-359 of the bovine CaR, which resides within the predicted amino-terminal extracellular domain of the CaR. The antiserum was subjected to further purification using an affinity column conjugated with the FF-7 peptide, and the affinity-purified antiserum was used for immunocytochemistry and Western blot analysis as described below. We have previously documented the specificity of this antiserum for the CaR (3). Each coverslip with adherent cells was washed once with phosphate-buffered saline (PBS), fixed with 4% formaldehyde in PBS for 5 min and washed with PBS once again. Fixed human monocytes were treated with DAKO Protein Block Serum-Free Solution (DAKO Corp., Carpenteria, CA) for 1 h and then incubated overnight at 47C with primary antiserum (anti-CaR antiserum 4637) at a concentration of 5 mg/ml in blocking solution (DAKO Corp.). Negative controls were carried out by performing the same procedure following preabsorption of the anti-CaR antiserum with 10 mg/ml of the synthetic CaR peptide against which it was raised. After washing the cells three times with 0.5% bovine serum albumin in PBS for 10 min, alkaline phosphatase (Alp)-coupled, goat anti-rabbit IgG (1:200; GIBCO/BRL) was added and incubated for 1 hour at room temperature. The cells were then washed three times with PBS for 10 min each, and the color reaction was developed for 10-20 min using a solution consisting of 44 ml Nitroblue Tetrazolium Chloride (NBT) (75 mg/ml) and 33 ml 5-Bromo-4-chloro-3-indolylphosphate p-Toluidine Salt (BCIP) (50 mg/ml) in 10 ml of 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2, and 1 mg/ml levamisole, which was included for inhibition of endogenous cellular Alp activity. The color reaction was stopped by washing twice in the above solution without NBT or BCIP and then twice in water. Staining for NSE. Adherent monocytes were stained for NSE using a kit in which the substrate for the enzyme is a-naphthyl acetate (Sigma Diagnostics, St. Louis, MO) according to the manufacturer’s instructions. Flow cytometric analysis. Flow cytometric analysis was performed using a dual laser FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA) calibrated by using 2 mm Calibrite beads (Becton Dickinson). Anti-CaR antiserum 4637 was added to peripheral blood mononuclear cells and incubated alone or with a CaR peptide for two hours at room temperature. Cells were washed with PBS containing 2% FCS, resuspended in PBS with 2% FCS and incubated for 30 min at room temperature with monoclonal Goat anti-rabbit IgG conjugated to R-phycoerythrin (Sigma). Cells were washed again in PBS with 2% FCS, resuspended and directly conjugated monoclonal antibody (CD 14-FITC, CD45-PERCP, Becton Dickinson) was added and incubated for 15 min at room temperature. Stained cells were fixed with 1% paraformaldehyde and assayed within 24 h. Data acquisition and analysis was performed on Macintosh Power PC (Apple Computer Inc.) using CellQuest software (Becton Dickinson). Western analysis of CaR in human peripheral blood monocytes. Adherent monocytes in a 25 cm2 culture flask were rinsed twice with 1 mM EDTA in PBS and lysed with 1.0 ml of a lysis solution (1% SDS, 10 mM Tris-HCl, pH 7.4) heated to 657C. The cells were scraped from the flasks, transferred to microcentrifuge tubes and heated for an additional 5 min at 657C. The viscosity of the sample was reduced by brief sonication, and insoluble material was removed by centrifugation for 5 min. The resultant whole cell lysate in the supernatant was stored at 0207C until Western blot analysis was carried out. Aliquots of 30 mg and 10 mg of proteins were dissolved in SDSLaemmli gel loading buffer with and without 100 mM dithiothreitol (DTT) to produce reducing and non-reducing conditions, respectively, incubated at 377C for 15 min and resolved electrophoretically on 6.5% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose at 100 V for 40 min in transfer buffer containing 19 mM Tris-HCl, 150 mM glycine, 0.015% SDS and 20% meth-
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anol. The blots were blocked for 2 h with 1% BSA in PBS containing 0.25% Triton X-100 (blocking solution) and then incubated overnight at 47C with the affinity-purified antiserum (4637) or with peptideblocked antiserum (the same amount of antiserum preincubated at room temperature for 60 min with twice the amount of FF-7 peptide) at a concentration of 1 mg/ml in the blocking solution. The blots were washed three times with PBS containing 0.25% Triton X-100 (washing solution) at room temperature for 10 min each. The blots were further incubated with a 1:2000 dilution of horseradish peroxidase-coupled, goat anti-rabbit IgG (Sigma Chemical Co.) in the blocking solution for 1 h at room temperature. The blots were then washed three times with the washing solution at room temperature for 40 min each, and specific protein bands were detected using an enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL). RT-PCR of CaR in human peripheral blood monocytes. Total RNA was prepared from adherent monocytes in a 25 cm2 culture flask with the TRIzol Reagent (GIBCO/BRL). 1 mg of total RNA was used for the synthesis of single stranded cDNA (cDNA synthesis kit, GIBCO/BRL). The resultant first-stranded cDNA was used for the PCR procedure. PCR was performed at a final concentration of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.8 mM MgCl2, 0.2 mM dNTP, 0.4 mM of forward primer, 0.4 mM of reverse primer and 1 ml of ELONGASE enzyme mix (a Taq/Pyrococcus species GB-D DNA polymerase mixture) (GIBCO/BRL). Human parathyroid CaR sense primer, 5*-CGGGGTACCTTAAGCACCTA-CGGCATCTAA-3*, and antisense primer, 5*-GCTCTAGAGTTAACGCGATCCCAAAGGGCTC-3*, were used for the reactions. This set of primers was designed to span two introns of the human CaR gene in order to avoid confusion arising from amplification of the same sequence within contaminating genomic DNA. In order to perform ‘hot start’ PCR, the enzyme was added during the initial 3-minute denaturation and was followed by 35 cycles of amplification (30-sec denaturation at 947C, 30-sec annealing at 477C, and 1-min extension at 727C). The reaction was completed with an additional 10-min incubation at 727C to allow completion of extension. PCR products were fractionated on 1.2% agarose gels. The presence of a 485 base pair (bp) amplified product was indicative of a positive PCR reaction arising from CaR-related sequence within cDNA. The PCR product in the reaction mixture was purified using the QIAquick PCR purification kit (Qiagen, Santa Clara, CA) and subjected to direct, bi-directional sequencing employing the same primer pairs used for PCR by means of an automated sequencer (AB377, Applied Biosystems, Foster City, CA) in the DNA Sequence Faculty of the University of Maine (Orono, ME), using dideoxy terminator Taq technology.
RESULTS Immunoreactivity of CaR protein in human peripheral blood monocytes using CaR-specific antiserum. We initially performed immunocytochemistry with a specific anti-CaR antiserum on human monocytes, which were isolated as cells that adhered to plastic following Histopaque fractionation of human blood. Figure 1A reveals strong CaR immunoreactivity on the cell surface and over the cytoplasm of monocytes, which was present in 93% of 161 cells counted and was eliminated by preincubating the primary antiserum with the peptide against which it was raised (Fig. 1B). The monocytic character of the cells was confirmed by their strong, homogeneous NSE staining, which was present in 89% of 266 cells counted, as well as by their characteristic cellular morphology (Fig. 1C). Low density peripheral blood mononuclear cells were also evaluated by flow cytometry. Cells staining with
FIG. 1. Immunocytochemistry of human peripheral blood monocytes carried out as described in Materials and Methods using a CaR-specific polyclonal antiserum (4637). (A) Immunocytochemistry revealed strong CaR immunoreactivity in human monocytes. (B) The staining was eliminated by preincubating the primary antiserum with the peptide against which it was raised. (C) The monocytic character of the cells was confirmed by their strong, homogeneous NSE staining. The photomicrographs were taken at a magnification of 1000X.
the monocyte marker CD14/CD45 were found to stain positively for CaR (83%) which was inhibited by CaR peptide (Fig. 2). In contrast, only 4% of the lymphocyte population stained positively for CaR (Fig. 2).
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with the specific peptide against which it was raised, under both non-reducing and reducing conditions (Fig. 3, lanes 3 and 4, respectively). These results were confirmed by performing immunocytochemistry and/or Western analysis on peripheral blood monocytes taken from three different healthy men. Detection of CaR mRNA in human peripheral blood monocytes by RT-PCR. RT-PCR with CaR-specific, intron-spanning primers, which were utilized to ensure that any products were not the result of amplification from contaminating genomic DNA, amplified a product of the size (485 bp) expected for a CaR-derived product from cDNA synthesized from total RNA isolated from human monocytes (Fig. 4). DNA sequence analysis of the PCR product revealed 100% homology with the human parathyroid CaR cDNA sequence (data not shown). DISCUSSION Our results show that human peripheral blood monocytes clearly express CaR protein as assessed by immunocytochemistry (Fig. 1) flow cytometry (Fig. 2), and Western blot analysis (Fig. 3). The latter revealed a specific band at a molecular weight consistent with that of the intact, glycosylated CaR (16). In addition, RTPCR performed on total RNA isolated from these cells followed by sequence analysis of the PCR product indi-
FIG. 2. CaR expression on monocytes and lymphocytes. Data were generated by using three color flow cytometry. Monocytes were gated based on their CD14/CD45 expression. The placement of histogram markers was determined by fluorescence intensity of cells pre-treated with CaR peptide and stained with polyclonal anti-CaR serum.
We also performed Western analysis on proteins isolated from human peripheral blood monocytes (Fig. 3). Under non-reducing conditions, a specific band at Ç170 kDa was identified that was of a size compatible with that of the intact, glycosylated human CaR (16) (lane 1). Under reducing conditions, a specific band at Ç60 kDa was prominent and likely represented a degradation product of the CaR, since the non-glycosylated CaR has an expected molecular weight of Ç120 kDa (16) (lane 2). The specificity of all of these bands was apparent from the marked diminution in their intensities following preabsorption of the anti-CaR antiserum
FIG. 3. Western analysis of whole cell lysates from human peripheral blood monocytes under non-reducing and reducing conditions (lanes 1 and 2, respectively), performed as described in Materials and Methods. Lane 1, a prominent band at Ç170 kDa that was stained in the presence of specific antiserum under non-reducing conditions was of a size consistent with that of the intact, glycosylated CaR. Lane 2, the band at Ç60 kDa may represent a degradation product of the CaR protein generated following reduction of the CaR by DTT treatment. The specificity of the labeling by the antiserum was confirmed by marked reduction in the intensities of the bands under both non-reducing and reducing conditions following preabsorption of the anti-CaR antiserum with the specific peptide against which it was raised (lanes 3 and 4, respectively).
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FIG. 4. Identification of CaR transcript in human peripheral blood monocytes by RT-PCR, performed as described in Materials and Methods. The product amplified by RT-PCR was of the expected size (485 bp) for having arisen from bona fide CaR transcript (right lane). DNA sequence analysis of the PCR product revealed 100% homology with the human parathyroid CaR cDNA sequence (data not shown). The 1 kb ladder (GIBCO/BRL) was used as a size marker (left lane).
cated the presence of bona fide CaR transcript (Fig. 4). Thus, although Bornefalk et al. failed to detect CaR transcript in human peripheral blood monocytes using RT-PCR (14), the present study ensures that these cells actually express both CaR protein and mRNA. When Western blot analysis was performed under reducing conditions (Fig. 3, lane 2), human monocytes showed a low molecular weight band that most likely represented a degradation product, perhaps because reduced forms of the CaR were more susceptible to endogenous proteases. We have previously found that degradation of the CaR following treatment with DTT was also apparent in other two human cell lines expressing the CaR (our unpublished observations), suggesting that such degradation occurs not uncommonly, particularly in cell types containing abundant protease activity, such as macrophage/monocytes. Therefore, the use of non-reducing conditions during Western blot analysis may facilitate identifying CaR immunoreactive bands at a molecular weight(s) consistent with the presence of the intact CaR, including its various glycosylated forms. Although peripheral blood monocytes might not be identical to the mononuclear cells present at sites of bone remodeling, accumulating evidence suggests that monocytes in the circulation, regardless of their site of origin, can represent a useful model of the functions of mononuclear cells localized at resorptive sites within bone (8-14). In addition, we have recently shown that NSE-positive mononuclear cells in primary cultures of mouse bone marrow also express CaR protein by immunocytochemistry (3). Since these monocyte-like cells were taken directly from bone marrow, they could po-
tentially be similar to those appearing at sites of osteoclastic bone resorption during the reversal phase of bone remodeling in vivo. This previous study, as well as the present one, strengthens the evidence that mononuclear cells localized at the resorptive site in bone marrow in vivo can express the CaR. Several studies by other researchers have suggested that human peripheral blood monocytes have a Cao2/sensing mechanism, which can directly stimulate the secretion of cytokines (14) and modulate their proliferation (11) as well as indirectly influence the proliferation and migration of osteoblastic cells (11, 12) and inhibit osteoclast formation (13) via humoral factors. These in vitro actions of elevated Cao2/ on monocytes may also occur in vivo at sites of bone resorption following the termination of the resorptive phase of bone remodeling and contribute to the initiation of bone formation (6). Clinically, secretion of cytokines from human peripheral blood monocytes is known to increase following the development of estrogen deficiency in the immediate postmenopausal period (5) and to reflect bone formation rates in osteoporotic patients (8). Taken together, available evidence suggests that this putative Cao2/sensing mechanism in human peripheral blood monocytes could potentially be of substantial physiological importance by virtue of being intimately involved in the bone remodeling process in vivo. In summary, our present results show that human monocytes express both CaR protein and mRNA, suggesting that the CaR may represent the molecular basis for the previously described Cao2/-sensing capacity of these cells. ACKNOWLEDGMENTS The authors gratefully acknowledge generous grant support from the following sources: the Yamanouchi Foundation Grant for Research on Metabolic Disorders (to T.Y.), the Mochida Memorial Foundation Grant for Medical and Pharmaceutical Research (to T.Y.), NPS Pharmaceuticals, Inc. (to E.M.B.), the St. Giles Foundation (to E.M.B.), the National Space Bioscience Research Institute (NSBRI) (to E.M.B.), the National Institutes of Health (DK 50234) (to D.T.S.), and the Richard Saltonstall Charitable Trust (to D.T.S.).
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7. Silver, I. A., Murrills, R. J., and Etherington, D. J. (1988) Exp. Cell. Res. 175, 266–276. 8. Pacifici, R., Rifas, L., Teitelbaum, S. L., Slatopolsky, E., McCracken, R., Bergfeld, M., Lee, W., Avioli, L. V., and Peck, W. A. (1987) Proc. Natl. Acad. Sci. USA 84, 4616–4620. 9. Pacifici, R., Carano, A., Santoro, S. A., Rifas, L., Jeffrey, J. J., Malone, J. D., McCracken, R., and Avioli, L. V. (1991) J. Clin. Invest. 87, 221–228. 10. Fujikawa, Y., Quinn, J. M. W., Sabokbar, A., McGee, J. O. D., and Athanasou, N. A. (1996) Endocrinology 137, 4058–4060. 11. Sugimoto, T., Kanatani, M., Kano, J., Kaji, H., Tsukamoto, T., Yamaguchi, T., Fukase, M., and Chihara, K. (1993) J. Bone Miner. Res. 8, 1445–1452.
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