0021-972X/90/7103-0675$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 3 Printed in U.S.A.
Calcitonin Receptors on Circulating Normal Human Lymphocytes* J. J. BODY, F. GLIBERT, S. NEJAI, G. FERNANDEZ, A. VAN LANGENDONCK, AND A. BORKOWSKI Service de Medecine et Laboratoire d'Investigation Clinique H. J. Tagnon, Bone Metabolism Unit, Institut Jules Bordet, 1 Rue Heger-Bordet, 1000 Bruxelles, Belgium
ABSTRACT. Circulating human lymphocytes possess specific and functional receptors for calcitriol and PTH. We sought to determine if they also possessed receptors for calcitonin (CT), the third classical calciotropic hormone. We isolated blood mononuclear cells from healthy volunteers to separate monocytes, total lymphocytes, and T-lymphocytes; the purity of the three cell populations was more than 90%, 95%, and 85%, respectively. Salmon CT (sCT) was labeled by the chloramineT method (SA, 254 /iCi/Vg) without loss of biological activity. We found saturable (16 h at 8 C), specific, high affinity binding sites for [125I]sCT on unstimulated lymphocytes. As for CT receptors on other cells, binding of [125I]sCT was poorly reversible. Binding specificity was demonstrated by the total absence of competing effect of several unrelated hormones; human CT and CT gene-related peptide competed much less efficiently than sCT for the binding sites, whereas PDN-21 had no effect. When plotted according to the method of Scatchard, binding
T
data on the mixed population of T- and B-lymphocytes showed an apparent Kd (mean ± SD) of 2.9 ± 1.0 x 10"10 M (n = 34), with an estimation of 91-8338 (median, 1971) binding sites/cell. The data were repeatedly compatible with an aspect of positive cooperativity between the binding sites, as confirmed by a Hill coefficient greater than 1 (1.18 ± 0.13). However, this aspect of positive cooperativity in CT binding was not observed on isolated T-lymphocytes (Hill coefficient, 0.96 ± 0.08; n = 9; P < 0.001 us. the mixed population of lymphocytes). CT did not induce a significant increase in cAMP levels, but regulation of receptor concentration was demonstrated by the finding of down-regulation of CT-binding sites after sCT or human CT preincubation. In summary, we have found saturable, specific, high affinity receptors for CT on unstimulated normal human T-lymphocytes, which could, thus, be target sites for CT action on bone metabolism or on the immune system. (J Clin Endocrinol Metab 7 1 : 675-681, 1990)
(4), and preliminary evidence suggests that PTH could stimulate their bone-resorbing activity (5). Whereas osteoclasts are well established target cells for calcitonin (CT) (6), CT receptors have also been described on various lymphoid cell lines (7). We report here the existence of specific CT plasma membrane receptors on circulating normal human lymphocytes. This observation further strengthens the idea of interactions between calciotropic hormones and immune cells and points to so far unrecognized target cells for the pharmacological and/or physiological actions of CT.
HE IMPORTANCE of lymphocytes and monocytes to the physiological control and pathology of bone metabolism is increasingly recognized. Whereas osteoclasts belong to the mononuclear-phagocyte system, lymphocytes and monocytes are also the source of several cytokines that potently stimulate bone resorption, such as tumor necrosis factor, lymphotoxin, and interleukin1, or that inhibit cytokine-mediated bone resorption, such as interferon-7 (1). On the other hand, circulating mononuclear cells possess specific and functional receptors for the two main calciotropic hormones. Indeed, calcitriol, the active form of vitamin D, specifically binds to monocytes and activated T-lymphocytes (2), and has antiproliferative effects on these cells, probably through inhibition of interleukin-2 and interferon-7 production (3). PTH receptors have also been found on human circulating lymphocytes
Materials and Methods Cell isolation We derived our isolation procedure of circulating lymphocytes and monocytes from the reports of Freundlich and Avdalovic (8) and Horton et al. (9). Cells were obtained from healthy individuals who provided written informed consent for blood withdrawal. Heparinized blood [100-150 mL/experiment; 10 U heparin (Calparin)/mL blood] was diluted 1:1 with phosphatebuffered saline (PBS, Gibco, Gent, Belgium) and layered onto 5% Ficoll-9.6% sodium metrizoate (density, 1.077 g/mL; Lymphoprep, Nycomed, Oslo, Norway). The tubes (30 mL diluted
Received July 5, 1989. Address all correspondence and requests for reprints to: Dr. J. J. Body, Service de Medecine et Laboratoire d'Investigation Clinique H. J. Tagnon, Bone Metabolism Unit, Institut Jules Bordet, 1 rue HegerBordet, 1000 Brussels, Belgium. * This work was supported by a grant from the Fondation Lefevre (Brussels, Belgium). 675
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BODY ET AL.
676
blood/15 mL Lymphoprep) were then centrifuged at a speed progressively reaching 1000 X g for 20 min at 18 C. Plasma was recovered, centrifuged (750 x g for 10 min at 15 C), and layered over petri flasks (3002 F, Falcon, Becton-Dickinson, Erembodegem, Belgium) previously coated with gelatin (0.2%); 4 mL plasma were deposited in each flask for 1 h at 37 C. During that time, the buffy coats collected at the interfaces were centrifuged at 750 x g for 10 min at 15 C; the cells were then put together, washed with PBS, and counted in a Neubauer cell (after centrifugation at 30 x g for 10 min at 15 C). The plasma-treated flasks were rinsed with calcium- and magnesium-free PBS before receiving the cells. We incubated 9 X 106 cells/flask for 1 h at 37 C in 3 mL complete medium, which consisted of RPMI-1640 (with 200 mM 2% L-glutamine, 10,000 U penicillin, and 10 mg streptomycin/mL) containing 15% inactivated horse serum and 5% fetal calf serum (FCS; all reagents from Gibco). After incubation, cells in the supernatants, mainly lymphocytes, were collected and thereafter mixed with the cells obtained by two subsequent washes of the flasks (3 mL RPMI-1640 containing 10% FCS). After further centrifugation and washing, the lymphocytes were diluted in complete medium before binding studies. The cell viability routinely exceeded 97% (as determined by the exclusion of trypan blue) and the purity 95% (as determined by light microscopy and esterase staining, and confirmed by cytofluorometry). Monocytes were recovered by treating the flasks with 2 mM EDTA in complete medium at 37 C (3 mL/flask for 10 min), with regular mild shaking of the flasks. The monocyte-containing supernatants were collected with a Pasteur pipet; the flasks were washed once with RPMI-1640 containing 10% FCS at 37 C. Monocytes were then put together and similarly diluted in complete medium; their viability exceeded 95% and their purity 90%. Lymphocyte and monocyte preparations were contaminated by less than 0.2% red blood cells and less than 0.1% platelets and polymorphonuclear cells. In some experiments we further separated T-lymphocytes from B-lymphocytes using the method of Mage and McHugh (10); 20 x 106 lymphocytes were incubated for 20 min at room temperature on flasks (Falcon 1029 F) previously coated with immunoglobulins (goat Ig antihuman IgG and human IgG). Nonadherent T-lymphocytes were further depleted of B-lymphocytes by two consecutive incubations on other IgG-coated flasks (10). The purity of T-lymphocytes was between 85-95% (as determined by cytofluorometry using a panel of fluorescent monoclonal antibodies). In some experiments requiring a large number of cells, mononuclear cells were isolated from buffy coats obtained from the Transfusion Unit of University Hospital Saint-Pierre (Brussels, Belgium). Iodination of CT Salmon CT (sCT; generously given by Sandoz S.A., Basel, Switzerland) was iodinated according to the method of Fischer et al. (11). In short, 2.5 Mg sCT were iodinated with Na125I (0.5 mCi) by chloramine-T (5.1 x 10"4 M); the reaction was stopped by the addition of metabisulfite (6 x 10"3 M) and plasma. Labeled sCT ([125I]sCT) was purified on a Bio-Gel P30 column. The mean specific activity was 254 nC\/n% (determined by self-
JCE & M • 1990 Vol 71 • No 3
displacement in a RIA system thanks to As G-281 generously given by Prof. J. Fischer, Zurich, Switzerland). The biological activity of sCT was not altered by the iodination procedure, as indicated by the results obtained in the in vivo bioassay of Sturtridge and Kumar (12): the mean (±SE) decreases in serum calcium (measured 1 h after sc CT injection) in groups of 5-12 young rats were, for example, 0.45 ± 0.01 mmol/L with 8 ng unlabeled sCT vs. 0.59 ± 0.04 mmol/L with 8 ng [125I]sCT, and 0.48 ± 0.04 mmol/L with 32 ng unlabeled sCT vs. 0.49 ± 0.03 mmol/L with 32 ng [125I]sCT (P = NS). We also verified that the chromatographic profile of [126I] sCT was not altered after incubation with lymphocytes (16 h at 8 C). Binding studies Freshly prepared cells (2 X 106/mL) were incubated with [ I]sCT (~10~10 M unless otherwise specified) in microtubes previously coated with BSA (0.3%; tubes were allowed to air dry before use). The composition of the incubation medium was adapted from the study by Marx and collaborators (7) as follows: 15 mM Tris, 120 mM NaCl, 4 mM KC1,1.6 mM MgSO4, 2 mM NaH2PO4,10 mM glucose, 0.1% human albumin, and 500 U/mL aprotinin (Trasylol). Incubation was terminated by centrifugation (2.5 min) in a Beckman microfuge (Beckman, Palo Alto, CA). The percent binding was calculated by counting (ycounter, Packard, Downers Grove, IL) first the total radioactivity, then, after centrifugation, supernatant decantation and precipitate washing, by counting the radioactivity contained in the cellular pellet obtained by cutting off the tips of the tubes. Each condition was duplicated between four and eight times, and nonspecific binding (NSB) was systematically determined by measuring the tracer bound to the cells in the presence of 5.25 X 10~6 M unlabeled sCT. The mean NSB was 11% of the total binding, and all results were expressed as specific binding (total binding observed in the presence of tracer only minus NSB). To control binding specificity, the competitive inhibition of [125I]sCT (±10~10 M) binding to lymphocytes was studied using varying concentrations of unlabeled sCT, human CT (hCT; a gift of Drs. Scheibli and Andreatta, Ciba-Geigy, Basel, Switzerland), glucagon (a gift of Novo Industria, Copenhagen, Denmark), ACTH-(l-39) (a gift of Organon, Oss, The Netherlands), CT gene-related peptide (CGRP), PDN-21 (also called katacalcin), insulin, and somatostatin (all latter peptides were of human origin and bought from Peninsula Laboratories, Belmont, CA). In some experiments, binding of [125I]sCT was measured after preincubation with high concentrations of unlabeled hormones. Cells were first cultured for 24 h in complete medium, then for another 24 h in fresh medium containing 10~7 M sCT, hCT, or CGRP; after extensive washing, the cells were incubated with [125I]sCT, as described above. The binding data were transformed according to the classical methods of Scatchard and Hill (13-15). 125
Measurement of circulating CT Plasma CT was measured using our previously described extraction procedure, followed by radioimmunological determination (16, 17).
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677
CT RECEPTORS ON HUMAN LYMPHOCYTES Membrane preparation Membranes were prepared from isolated lymphocytes according to the method of Morimoto et al. (18). Measurement of cAMP production Adenylate cyclase activity was evaluated by the measurement of cAMP production [measured by an in-house RIA (19), using the As G-828 given by Dr. H. Heath III, Rochester, MN] at various time intervals after the addition of CT. Experiments were carried out on intact cells (measurement of extra- and intracellular cAMP) and on membrane preparations, with 2.2 mM isobutylmethylxanthine (Sigma, St. Louis, MO) in the incubation medium to inhibit phosphodiesterase. IBMX was present in the incubation medium 20 min before the addition of CT or prostaglandin E2 (PGE2), which served as a positive control.
Results Evaluation of CT binding to monocytes CT binding to monocytes was low, not saturable, and not proportional to the cell number, which strongly argues against the presence of specific CT-binding sites on circulating adult monocytes (data not shown). All subsequent experiments were, therefore, conducted on isolated unstimulated lymphocytes.
more rapid at 20 C, since maximal binding was obtained within 4 h and remained constant for at least another 4 h. Tracer degradation at 37 C prevented a reliable determination of optimal conditions for CT binding at this temperature. All subsequent experiments were performed at 8 C for 16 h, and under these conditions, tracer integrity, as assessed by 20% trichloroacetic acid precipitation, was greater than 85%. Chromatographic studies also showed that [125I]sCT was not degraded during cell incubation. Binding was linearly related to cell density at least up to 4 X 106 cells/mL (data not shown). CT-binding sites were saturable, as shown in Fig. 2. The percentage of [125I]sCT binding varied between 0.2513.1% (median, 3.0%; n = 64); only 14% (4-38%) of the binding was nonspecific. There was no detectable influence of sex or age of the donors on the percentage of specific [125I]sCT binding on lymphocytes. As shown in Fig. 3, CT binding to lymphocytes was poorly reversible. The time course of dissociation of radioligand was assessed with or without an excess of unlabeled CT up to 72 h after stopping the incubation; the dissociation half-time was between 3-12 hours, but the dissociation was never complete and was only minimally influenced by the presence of excess unlabeled hormone. Chromatographic studies of the medium after a 72-h incubation suggested that most of the radioactivity still comigrated with [125I]sCT (not shown).
Characteristics of CT binding to lymphocytes The time course of CT binding to lymphocytes was studied at 8 and 20 C to determine optimal binding conditions. At 8 C, specific binding reached a plateau within 12 h and was stable for at least another 8 h (see Fig. 1). An initial plateau after 1-2 h was frequently observed and is depicted in the inset of Fig. 1. This first plateau has not been systematically investigated because it occurred at a very low specific binding. Binding was
Competitive binding of CT to lymphocytes A dose-dependent decrease in the specific binding of [125I]sCT was observed in the presence of increasing concentrations of unlabeled sCT under steady state conditions (Fig. 4). The IC50 value was between lO^-lO" 10 M and was not significantly influenced by a delayed 0.5
2.0
bind
5S
1
0.4
S x S
1.5
m
1.0
0.3
0.2
0.1 0.5
0.0
80 0.0
6
8
10
12
14
16
18
10
T.nMxiO" 20
Time, hours
FIG. 1. Time course of CT binding to lymphocytes. [125I]sCT (10~10 M) was incubated with 2 x 106 cells/mL at 8 C ( • - • ) during the indicated time intervals. Inset, Analysis of the first 90 min.
30
20
40
2
FIG. 2. Saturability of CT binding to lymphocytes. Cells (2 x 106/mL) were incubated at 8 C with varying concentrations of [125I]sCT (T). [125I]sCT binding (B) was determined after 16 h. NSB was determined in concurrent incubations with 5.25 X 10~6 M unlabeled sCT. Specific binding (B) represents the difference between total (T) and NSB. AA, Total binding; • - • , specific binding; O-O, NSB.
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BODY ET AL.
678 100-, 80-
I 60 "3 4020-
16
24
32 40 Time, hours
48
56
64
72
FIG. 3. Dissociation of CT binding from lymphocytes. Cells were incubated with [125I]sCT for 16 h at 8 C, centrifuged, washed, and resuspended in fresh incubation medium with ( • - • ) or without (x- -x) unlabeled sCT (5 x 10~6 M). Binding of [125I]sCT was then determined at the indicated time intervals in different incubation tubes. The data are expressed as percentages of initial specific binding.
Hormone concentration, log FlG. 4. Specificity of CT binding to lymphocytes. Competitive inhibition of the binding of [125I]sCT was evaluated for the following peptides: sCT (•), CGRP (O), hCT (A), PDN-21 (•), insulin (•), glucagon ACTH-(l-39) and somatostatin (A). Data are expressed as percentages of specific binding without competitor, and for sCT, are the mean ± SE of five separate experiments.
tracer addition or when competition experiments were performed on isolated T-lymphocytes (data not shown). CGRP and hCT were 100-1000 times less effective than sCT, whereas PDN-21 (katacalcin), insulin, glucagon, ACTH-(l-39), or somatostatin did not inhibit the binding of [125I]sCT (Fig. 4).
JCE & M • 1990 Vol 71 • No 3
Scatchard plot with the abscissa, was 1971 (n = 34; range, 98-8338), with a mean (±SD) apparent Kd of 2.9 ± 1.0 X 10~10 M. The mean Hill coefficient, a quantitative estimation of positive cooperativity, was 1.18 ± 0.13. The reliability of the determination of Hill coefficients was quite satisfactory: when measured at two or three different times in four individuals, the figures did not differ by more than 3% for the same person. To further investigate this aspect of positive cooperativity between CT-binding sites, we first demonstrated that it was apparently not due to artefactual factors. There was, indeed, no degradation or polymerization of the radioligand, and we also excluded NSB of the radiotracer to a substance in the incubation medium. This was demonstrated by control chromatographic profiles (on Bio-Gel P-3O and P-1OO) of low quantities of [125I]
sCT incubated with lymphocytes. Moreover, in the same experimental conditions, positive cooperativity was no longer observed when CT binding was determined on isolated T-lymphocytes, as shown in Fig. 6 by a representative study. The Scatchard analysis then revealed a single class of high affinity binding sites with an apparent Kd of 1.8 ± 1.3 X 10"10 M (n = 9) and 415-4666 (median, 2020) binding sites/cell. The mean Hill coefficient was 0.96 ± 0.08, which was significantly less (P < 0.001) than the mean Hill coefficient calculated for experiments performed on the mixed population of T- and B-lymphocytes. Moreover, to determine if a soluble factor from Blymphocytes or from the few contaminating monocytes was responsible for this phenomenon of positive cooperativity, we determined binding parameters in three experiments simultaneously performed with cells isolated from the same pool: mixed T- and B-lymphocytes, isolated T-lymphocytes, and isolated T-lymphocytes incubated for 24 h in conditioned medium from B- and Tlymphocytes. The apparent Kd values were similar (~10~10 M) for the three experiments, but the Hill coefficients were 1.12,1.01, and 1.17, respectively, suggesting restoration of the positive cooperativity between binding sites. Binding of [125I]sCT to B-lymphocytes was negligible or very low (
Vol. 71, No. 3 Printed in U.S.A.
Calcitonin Receptors on Circulating Normal Human Lymphocytes* J. J. BODY, F. GLIBERT, S. NEJAI, G. FERNANDEZ, A. VAN LANGENDONCK, AND A. BORKOWSKI Service de Medecine et Laboratoire d'Investigation Clinique H. J. Tagnon, Bone Metabolism Unit, Institut Jules Bordet, 1 Rue Heger-Bordet, 1000 Bruxelles, Belgium
ABSTRACT. Circulating human lymphocytes possess specific and functional receptors for calcitriol and PTH. We sought to determine if they also possessed receptors for calcitonin (CT), the third classical calciotropic hormone. We isolated blood mononuclear cells from healthy volunteers to separate monocytes, total lymphocytes, and T-lymphocytes; the purity of the three cell populations was more than 90%, 95%, and 85%, respectively. Salmon CT (sCT) was labeled by the chloramineT method (SA, 254 /iCi/Vg) without loss of biological activity. We found saturable (16 h at 8 C), specific, high affinity binding sites for [125I]sCT on unstimulated lymphocytes. As for CT receptors on other cells, binding of [125I]sCT was poorly reversible. Binding specificity was demonstrated by the total absence of competing effect of several unrelated hormones; human CT and CT gene-related peptide competed much less efficiently than sCT for the binding sites, whereas PDN-21 had no effect. When plotted according to the method of Scatchard, binding
T
data on the mixed population of T- and B-lymphocytes showed an apparent Kd (mean ± SD) of 2.9 ± 1.0 x 10"10 M (n = 34), with an estimation of 91-8338 (median, 1971) binding sites/cell. The data were repeatedly compatible with an aspect of positive cooperativity between the binding sites, as confirmed by a Hill coefficient greater than 1 (1.18 ± 0.13). However, this aspect of positive cooperativity in CT binding was not observed on isolated T-lymphocytes (Hill coefficient, 0.96 ± 0.08; n = 9; P < 0.001 us. the mixed population of lymphocytes). CT did not induce a significant increase in cAMP levels, but regulation of receptor concentration was demonstrated by the finding of down-regulation of CT-binding sites after sCT or human CT preincubation. In summary, we have found saturable, specific, high affinity receptors for CT on unstimulated normal human T-lymphocytes, which could, thus, be target sites for CT action on bone metabolism or on the immune system. (J Clin Endocrinol Metab 7 1 : 675-681, 1990)
(4), and preliminary evidence suggests that PTH could stimulate their bone-resorbing activity (5). Whereas osteoclasts are well established target cells for calcitonin (CT) (6), CT receptors have also been described on various lymphoid cell lines (7). We report here the existence of specific CT plasma membrane receptors on circulating normal human lymphocytes. This observation further strengthens the idea of interactions between calciotropic hormones and immune cells and points to so far unrecognized target cells for the pharmacological and/or physiological actions of CT.
HE IMPORTANCE of lymphocytes and monocytes to the physiological control and pathology of bone metabolism is increasingly recognized. Whereas osteoclasts belong to the mononuclear-phagocyte system, lymphocytes and monocytes are also the source of several cytokines that potently stimulate bone resorption, such as tumor necrosis factor, lymphotoxin, and interleukin1, or that inhibit cytokine-mediated bone resorption, such as interferon-7 (1). On the other hand, circulating mononuclear cells possess specific and functional receptors for the two main calciotropic hormones. Indeed, calcitriol, the active form of vitamin D, specifically binds to monocytes and activated T-lymphocytes (2), and has antiproliferative effects on these cells, probably through inhibition of interleukin-2 and interferon-7 production (3). PTH receptors have also been found on human circulating lymphocytes
Materials and Methods Cell isolation We derived our isolation procedure of circulating lymphocytes and monocytes from the reports of Freundlich and Avdalovic (8) and Horton et al. (9). Cells were obtained from healthy individuals who provided written informed consent for blood withdrawal. Heparinized blood [100-150 mL/experiment; 10 U heparin (Calparin)/mL blood] was diluted 1:1 with phosphatebuffered saline (PBS, Gibco, Gent, Belgium) and layered onto 5% Ficoll-9.6% sodium metrizoate (density, 1.077 g/mL; Lymphoprep, Nycomed, Oslo, Norway). The tubes (30 mL diluted
Received July 5, 1989. Address all correspondence and requests for reprints to: Dr. J. J. Body, Service de Medecine et Laboratoire d'Investigation Clinique H. J. Tagnon, Bone Metabolism Unit, Institut Jules Bordet, 1 rue HegerBordet, 1000 Brussels, Belgium. * This work was supported by a grant from the Fondation Lefevre (Brussels, Belgium). 675
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676
blood/15 mL Lymphoprep) were then centrifuged at a speed progressively reaching 1000 X g for 20 min at 18 C. Plasma was recovered, centrifuged (750 x g for 10 min at 15 C), and layered over petri flasks (3002 F, Falcon, Becton-Dickinson, Erembodegem, Belgium) previously coated with gelatin (0.2%); 4 mL plasma were deposited in each flask for 1 h at 37 C. During that time, the buffy coats collected at the interfaces were centrifuged at 750 x g for 10 min at 15 C; the cells were then put together, washed with PBS, and counted in a Neubauer cell (after centrifugation at 30 x g for 10 min at 15 C). The plasma-treated flasks were rinsed with calcium- and magnesium-free PBS before receiving the cells. We incubated 9 X 106 cells/flask for 1 h at 37 C in 3 mL complete medium, which consisted of RPMI-1640 (with 200 mM 2% L-glutamine, 10,000 U penicillin, and 10 mg streptomycin/mL) containing 15% inactivated horse serum and 5% fetal calf serum (FCS; all reagents from Gibco). After incubation, cells in the supernatants, mainly lymphocytes, were collected and thereafter mixed with the cells obtained by two subsequent washes of the flasks (3 mL RPMI-1640 containing 10% FCS). After further centrifugation and washing, the lymphocytes were diluted in complete medium before binding studies. The cell viability routinely exceeded 97% (as determined by the exclusion of trypan blue) and the purity 95% (as determined by light microscopy and esterase staining, and confirmed by cytofluorometry). Monocytes were recovered by treating the flasks with 2 mM EDTA in complete medium at 37 C (3 mL/flask for 10 min), with regular mild shaking of the flasks. The monocyte-containing supernatants were collected with a Pasteur pipet; the flasks were washed once with RPMI-1640 containing 10% FCS at 37 C. Monocytes were then put together and similarly diluted in complete medium; their viability exceeded 95% and their purity 90%. Lymphocyte and monocyte preparations were contaminated by less than 0.2% red blood cells and less than 0.1% platelets and polymorphonuclear cells. In some experiments we further separated T-lymphocytes from B-lymphocytes using the method of Mage and McHugh (10); 20 x 106 lymphocytes were incubated for 20 min at room temperature on flasks (Falcon 1029 F) previously coated with immunoglobulins (goat Ig antihuman IgG and human IgG). Nonadherent T-lymphocytes were further depleted of B-lymphocytes by two consecutive incubations on other IgG-coated flasks (10). The purity of T-lymphocytes was between 85-95% (as determined by cytofluorometry using a panel of fluorescent monoclonal antibodies). In some experiments requiring a large number of cells, mononuclear cells were isolated from buffy coats obtained from the Transfusion Unit of University Hospital Saint-Pierre (Brussels, Belgium). Iodination of CT Salmon CT (sCT; generously given by Sandoz S.A., Basel, Switzerland) was iodinated according to the method of Fischer et al. (11). In short, 2.5 Mg sCT were iodinated with Na125I (0.5 mCi) by chloramine-T (5.1 x 10"4 M); the reaction was stopped by the addition of metabisulfite (6 x 10"3 M) and plasma. Labeled sCT ([125I]sCT) was purified on a Bio-Gel P30 column. The mean specific activity was 254 nC\/n% (determined by self-
JCE & M • 1990 Vol 71 • No 3
displacement in a RIA system thanks to As G-281 generously given by Prof. J. Fischer, Zurich, Switzerland). The biological activity of sCT was not altered by the iodination procedure, as indicated by the results obtained in the in vivo bioassay of Sturtridge and Kumar (12): the mean (±SE) decreases in serum calcium (measured 1 h after sc CT injection) in groups of 5-12 young rats were, for example, 0.45 ± 0.01 mmol/L with 8 ng unlabeled sCT vs. 0.59 ± 0.04 mmol/L with 8 ng [125I]sCT, and 0.48 ± 0.04 mmol/L with 32 ng unlabeled sCT vs. 0.49 ± 0.03 mmol/L with 32 ng [125I]sCT (P = NS). We also verified that the chromatographic profile of [126I] sCT was not altered after incubation with lymphocytes (16 h at 8 C). Binding studies Freshly prepared cells (2 X 106/mL) were incubated with [ I]sCT (~10~10 M unless otherwise specified) in microtubes previously coated with BSA (0.3%; tubes were allowed to air dry before use). The composition of the incubation medium was adapted from the study by Marx and collaborators (7) as follows: 15 mM Tris, 120 mM NaCl, 4 mM KC1,1.6 mM MgSO4, 2 mM NaH2PO4,10 mM glucose, 0.1% human albumin, and 500 U/mL aprotinin (Trasylol). Incubation was terminated by centrifugation (2.5 min) in a Beckman microfuge (Beckman, Palo Alto, CA). The percent binding was calculated by counting (ycounter, Packard, Downers Grove, IL) first the total radioactivity, then, after centrifugation, supernatant decantation and precipitate washing, by counting the radioactivity contained in the cellular pellet obtained by cutting off the tips of the tubes. Each condition was duplicated between four and eight times, and nonspecific binding (NSB) was systematically determined by measuring the tracer bound to the cells in the presence of 5.25 X 10~6 M unlabeled sCT. The mean NSB was 11% of the total binding, and all results were expressed as specific binding (total binding observed in the presence of tracer only minus NSB). To control binding specificity, the competitive inhibition of [125I]sCT (±10~10 M) binding to lymphocytes was studied using varying concentrations of unlabeled sCT, human CT (hCT; a gift of Drs. Scheibli and Andreatta, Ciba-Geigy, Basel, Switzerland), glucagon (a gift of Novo Industria, Copenhagen, Denmark), ACTH-(l-39) (a gift of Organon, Oss, The Netherlands), CT gene-related peptide (CGRP), PDN-21 (also called katacalcin), insulin, and somatostatin (all latter peptides were of human origin and bought from Peninsula Laboratories, Belmont, CA). In some experiments, binding of [125I]sCT was measured after preincubation with high concentrations of unlabeled hormones. Cells were first cultured for 24 h in complete medium, then for another 24 h in fresh medium containing 10~7 M sCT, hCT, or CGRP; after extensive washing, the cells were incubated with [125I]sCT, as described above. The binding data were transformed according to the classical methods of Scatchard and Hill (13-15). 125
Measurement of circulating CT Plasma CT was measured using our previously described extraction procedure, followed by radioimmunological determination (16, 17).
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677
CT RECEPTORS ON HUMAN LYMPHOCYTES Membrane preparation Membranes were prepared from isolated lymphocytes according to the method of Morimoto et al. (18). Measurement of cAMP production Adenylate cyclase activity was evaluated by the measurement of cAMP production [measured by an in-house RIA (19), using the As G-828 given by Dr. H. Heath III, Rochester, MN] at various time intervals after the addition of CT. Experiments were carried out on intact cells (measurement of extra- and intracellular cAMP) and on membrane preparations, with 2.2 mM isobutylmethylxanthine (Sigma, St. Louis, MO) in the incubation medium to inhibit phosphodiesterase. IBMX was present in the incubation medium 20 min before the addition of CT or prostaglandin E2 (PGE2), which served as a positive control.
Results Evaluation of CT binding to monocytes CT binding to monocytes was low, not saturable, and not proportional to the cell number, which strongly argues against the presence of specific CT-binding sites on circulating adult monocytes (data not shown). All subsequent experiments were, therefore, conducted on isolated unstimulated lymphocytes.
more rapid at 20 C, since maximal binding was obtained within 4 h and remained constant for at least another 4 h. Tracer degradation at 37 C prevented a reliable determination of optimal conditions for CT binding at this temperature. All subsequent experiments were performed at 8 C for 16 h, and under these conditions, tracer integrity, as assessed by 20% trichloroacetic acid precipitation, was greater than 85%. Chromatographic studies also showed that [125I]sCT was not degraded during cell incubation. Binding was linearly related to cell density at least up to 4 X 106 cells/mL (data not shown). CT-binding sites were saturable, as shown in Fig. 2. The percentage of [125I]sCT binding varied between 0.2513.1% (median, 3.0%; n = 64); only 14% (4-38%) of the binding was nonspecific. There was no detectable influence of sex or age of the donors on the percentage of specific [125I]sCT binding on lymphocytes. As shown in Fig. 3, CT binding to lymphocytes was poorly reversible. The time course of dissociation of radioligand was assessed with or without an excess of unlabeled CT up to 72 h after stopping the incubation; the dissociation half-time was between 3-12 hours, but the dissociation was never complete and was only minimally influenced by the presence of excess unlabeled hormone. Chromatographic studies of the medium after a 72-h incubation suggested that most of the radioactivity still comigrated with [125I]sCT (not shown).
Characteristics of CT binding to lymphocytes The time course of CT binding to lymphocytes was studied at 8 and 20 C to determine optimal binding conditions. At 8 C, specific binding reached a plateau within 12 h and was stable for at least another 8 h (see Fig. 1). An initial plateau after 1-2 h was frequently observed and is depicted in the inset of Fig. 1. This first plateau has not been systematically investigated because it occurred at a very low specific binding. Binding was
Competitive binding of CT to lymphocytes A dose-dependent decrease in the specific binding of [125I]sCT was observed in the presence of increasing concentrations of unlabeled sCT under steady state conditions (Fig. 4). The IC50 value was between lO^-lO" 10 M and was not significantly influenced by a delayed 0.5
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FIG. 1. Time course of CT binding to lymphocytes. [125I]sCT (10~10 M) was incubated with 2 x 106 cells/mL at 8 C ( • - • ) during the indicated time intervals. Inset, Analysis of the first 90 min.
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FIG. 2. Saturability of CT binding to lymphocytes. Cells (2 x 106/mL) were incubated at 8 C with varying concentrations of [125I]sCT (T). [125I]sCT binding (B) was determined after 16 h. NSB was determined in concurrent incubations with 5.25 X 10~6 M unlabeled sCT. Specific binding (B) represents the difference between total (T) and NSB. AA, Total binding; • - • , specific binding; O-O, NSB.
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BODY ET AL.
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FIG. 3. Dissociation of CT binding from lymphocytes. Cells were incubated with [125I]sCT for 16 h at 8 C, centrifuged, washed, and resuspended in fresh incubation medium with ( • - • ) or without (x- -x) unlabeled sCT (5 x 10~6 M). Binding of [125I]sCT was then determined at the indicated time intervals in different incubation tubes. The data are expressed as percentages of initial specific binding.
Hormone concentration, log FlG. 4. Specificity of CT binding to lymphocytes. Competitive inhibition of the binding of [125I]sCT was evaluated for the following peptides: sCT (•), CGRP (O), hCT (A), PDN-21 (•), insulin (•), glucagon ACTH-(l-39) and somatostatin (A). Data are expressed as percentages of specific binding without competitor, and for sCT, are the mean ± SE of five separate experiments.
tracer addition or when competition experiments were performed on isolated T-lymphocytes (data not shown). CGRP and hCT were 100-1000 times less effective than sCT, whereas PDN-21 (katacalcin), insulin, glucagon, ACTH-(l-39), or somatostatin did not inhibit the binding of [125I]sCT (Fig. 4).
JCE & M • 1990 Vol 71 • No 3
Scatchard plot with the abscissa, was 1971 (n = 34; range, 98-8338), with a mean (±SD) apparent Kd of 2.9 ± 1.0 X 10~10 M. The mean Hill coefficient, a quantitative estimation of positive cooperativity, was 1.18 ± 0.13. The reliability of the determination of Hill coefficients was quite satisfactory: when measured at two or three different times in four individuals, the figures did not differ by more than 3% for the same person. To further investigate this aspect of positive cooperativity between CT-binding sites, we first demonstrated that it was apparently not due to artefactual factors. There was, indeed, no degradation or polymerization of the radioligand, and we also excluded NSB of the radiotracer to a substance in the incubation medium. This was demonstrated by control chromatographic profiles (on Bio-Gel P-3O and P-1OO) of low quantities of [125I]
sCT incubated with lymphocytes. Moreover, in the same experimental conditions, positive cooperativity was no longer observed when CT binding was determined on isolated T-lymphocytes, as shown in Fig. 6 by a representative study. The Scatchard analysis then revealed a single class of high affinity binding sites with an apparent Kd of 1.8 ± 1.3 X 10"10 M (n = 9) and 415-4666 (median, 2020) binding sites/cell. The mean Hill coefficient was 0.96 ± 0.08, which was significantly less (P < 0.001) than the mean Hill coefficient calculated for experiments performed on the mixed population of T- and B-lymphocytes. Moreover, to determine if a soluble factor from Blymphocytes or from the few contaminating monocytes was responsible for this phenomenon of positive cooperativity, we determined binding parameters in three experiments simultaneously performed with cells isolated from the same pool: mixed T- and B-lymphocytes, isolated T-lymphocytes, and isolated T-lymphocytes incubated for 24 h in conditioned medium from B- and Tlymphocytes. The apparent Kd values were similar (~10~10 M) for the three experiments, but the Hill coefficients were 1.12,1.01, and 1.17, respectively, suggesting restoration of the positive cooperativity between binding sites. Binding of [125I]sCT to B-lymphocytes was negligible or very low (
