Molecular and Cellular Endocrinology, 1 (1911) 19-81 0 Elsevier/North-Holland Scientific Publishers, Ltd.
THYROTROPIN-SPECIFIC BINDING TO HUMAN PERIPHERAL MONOCYTES AND POLYMORPHONUCLEAR LEUKOCYTES
BLOOD
Odile CHABAUD and Serge LISSITZKY Laboratoire de Biochimie M6dicale et U38 de 1‘INSERM, Facultt de Mdecine, Moulin, 13385 Marseille Cedex 4, France
Received 19 July 1916;accepted
27 Bd Jean-
14 September 1916
[ *251]Labeled thyrotropin binding to leukocytes has been studied using lymphocyte-, monocyte- and polymorphonuclear leukocyte-enriched preparations obtained by centrifugation on Ficoh-Angiocontrix gradients and Sephadex G-10 adherence. From the relation between thyrotropin binding and phagocytosis as shown by latex beads ingestion, it is concluded that the hormone binds essentially to monocytes and polymorphonuclear leukocytes. Equilibrium association constants of the high-affinity, low-capacity sites (1 nM_l) are similar to those found in isolated thyroid cells or in thyroid plasma membranes. The role of thyrotropin in the regulation of phagocytosis by leukocytes is discussed. Keywords:
thyrotropin;
monocytes; polymorphonuclear
leukocytes; phagocytosis.
It is well established that thyrotropin (TSH), the physiological thyroid stimulator, binds to specific receptor sites located at the external surface of the plasma membrane of the follicular thyroid cell. In addition to its action on the thyroid cell, thyrotropin can also modulate some activities in other tissues. The hormone increases lipolysis in rat and guinea pig adipose tissue (White and Engel, 1958) and recently thyrotropin receptor sites were described in purified plasma membranes from human and guinea pig adipocytes (Mullin et al., 1976). Also, thyrotropin or an associated pituitary factor was shown to decrease iodide metabolism in human polymorphonuclear leukocytes (Stoic, 1972). In the thyroid, the earliest effect of thyrotropin is the activation of adenylate cyclase (Pastan and Katzen, 1967; Yamashita and Field, 1970; Wolff and Jones, 1971; Verrier et al., 1974) a plasma membrane enzyme. In polymorphonuclear leukocytes, an increase in intracellular cyclic AMP was observed after addition of thyrotropin or prostaglandin Et (Stoic, 1972, 1974) suggesting that gra?ulocytes may contain receptor sites specific to thyrotropin. In the present communication, we describe the presence of thyrotropin-specific receptor sites in human leukocytes and especially in monocytes and polymorphonuclear leukocytes. 19
80
MATERIAL
0. Chabaud, S. Lissitzky
AND METHODS
Materials Sephadex G-10 and Ficoll were obtained from Pharmacia (Uppsala, Sweden) and Angiocontrix 48 from Laboratories Guerbet (Aulnay-sous-Bois, France). Purified porcine thyrotropin (20 U/mg in the McKenzie bioassay in the mouse) was kindly donated by Dr G. Hennen (LiBge). CeZZpreparations All the media and glasswares were sterilized before use. Human peripheral blood (100-l 50 ml) obtained from normal volunteers was collected on heparin. After dilution (1 : 1) with phosphate-buffered saline, pH 7.2, fractionation was performed on Ficoll [6.2% (w/v)] -Angiocontrix [ 11.4? (v/v)] gradients (d = 1.077 g/ml) according to the method of English and Andersen (1974). Centrifugation was for 20 min at 400g at the barrier in a swinging rotor. The interfaces were collected, pooled and the cells were washed twice and counted. Polymorphonuclear leukocytes were obtained using an additional layer of Ficoll-Angiocontrix (d = 1 .I2 g/ml). Viability as shown by exclusion of erythrosin B (0.1%) was on the average about 95%. Cells were suspensed in the appropriate assay medium and were stored at 0°C until utilization. Cell identification Lymphocytes, monocytes and polymorphonuclear leukocytes were identified by morphological criteria after staining with May-GriinewaldGiemsa (MGG) stain. Monocytes and polymorphonuclear leukocytes were also characterized by their aptitude to ingest latex beads (0.8 pm; 7.4 X lo8 per assay). Assays were performed for 30-45 min at 37°C in the medium used for [l*‘I]TSH binding experiments without EDTA and in the presence of 20% calf serum according to Cline and Lehrer (1968). Phagocytosis was monitored by microscopic examination of leukocyte smears stained by MGG or directly under phase-contrast microscopy. This was done by retention on SephaSeparation of lymphocytes and monocytes dex G-10 as proposed by Ly and Mishell (1974) for the separation of spleen cells. Monocyte-enriched preparations were obtained from the Sephadex G-10 columns after removal of the nonadherent cells and vortexing the Sephadex in excess medium as indicated by Schwartz et al: (1975). The yield in nonadherent lymphocytes was 56% of the cells put on the column and that in monocytes detached from the gel about 15%. Binding of (l*‘I/ TSH Porcine TSH was labeled with “‘1 using the lactoperoxidase method of iodide oxidation to a specific radioactivity of 60 @i/pg (Jaquet et al., 1974). Binding assays were performed in 25 mM Tris-Cl, 120 mM NaCl, 1.2 mM MgS04, 5 mM KCl, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, pH 7.4, containing 1 mg/ml bovine serum albumin (Archer et al., 1973). Cells were
[i25i]Thyrotropin
binding to leukocytes
81
suspended in the binding medium at a concentration of 15-50 X 1O6 cells/ml after isolation from Ficofl-Angiocontrix gradients and at a concentration of 4-15 X IO6 cells/ml after separation on Sephadex G-l 0. Binding assays were performed in I X 7 cm polyethylene tubes in 100 or 200 d final voiume at 24’C using 20-25 ng [lz51] TSH per ml. After the appropriate time of incubation, 4 ml of cold medium was added, the cells were separated by centrifugation as previously described (Lissitzky et al., 1973) and cell-associated radioactivity was counted. Specific binding was obtained by subtracting from the total radioactivity bound the amount which was not displaced by an excess of unlabeled TSH (1 PM). The experimental points in figures correspond to closely agreeing duplicates. Typical experiments are shown.
RESULTS Ceil preparations In 25 experiments, the average yield of cells obtained by Ficoll-Angiocontrix gradients (c! = 1.077 g/ml) (fraction L) was 1.2 f 0.074 X lo6 cells/ml blood. This fraction contained 86.1 + 2.3% lymphocytes, 10.6 + 1.6% monocytes and 1.1 + 0.1% polymorphonculear leukocytes. As shown by latex particle ingestion, the number of phagocytic cells was 18.0 + 2.1%. Platelets were in very low amount and red blood cells practically absent. After filtration on Sepahdex G-10, nonadherent cells contained 2.1 f 0.3% (MGG determination) and 4.9 + 1.8% (latex bead ingestion) monocytes. Adherent cehs (fraction M) released by vortexing of the Sephadex contained 12.8 + 3.0% (MGG determination) and 22.0 + 4.8% (latex bead ingestion) monocytes. Agreement between the two methods of monocyte estimation is not very good. Since identification by morphological criteria is somewhat difficult, characterization by phagocytic properties is likely to be quantitatively more reliable, contamination by polymorphonuclear leukocytes being less than 1%. The fractions enriched in polymorphonuclear leukocytes (fraction P) contained about 88% of these cells of which 71% were able to ingest latex beads. Contamination by monocytes was about 4% and varied for Iymphocytes between 4 and 10%. Binding of [ ’ 2 51Jthyro tropin fraction Binding of [ 12’ I] TSH was initially assayed on the lymphocyte-enriched L. The time-course of hormone binding (fig. 1) showed that equilibrium between association and dissociation was obtained in about 4.5 min. In 13 experiments the average specific TSH binding was 0.033 + 0.003% of the 0.9 nM [12’1] TSH added to the cells. This very low binding capacity suggested that all the cells present in the preparation were not able to bind the hormone. On the basis of the observations of Stoic (1971, 1972a), on the inhibitory effect of TSH on iodine metabolism in polymorphonuclear leukocytes and those of Schwartz et al. (1975) on the bind-
82
Fig. 1. Time-course
0. Chabaud, S. Lissitzky
of specific
CYtes,2 experiments); 25-40
[ ‘25I] TSH binding to leukocytes (fraction L: 4 and 6% monoX lo6 cells/ml were incubated at 24°C in the presence of 0.9 nm
[‘251]TSH.
ing of insulin to monocytes, it is possible that these cells might be responsible for TSH binding. This was ascertained using monocyte-enriched preparations of mononuclear leukocytes. Table 1 shows that when the percentage of monocytes as shown by their phagocytic property was increased from about 4% to about 20% by passage over Sephadex G-10 columns, a 3-fold increase in TSH specific binding was observed. In addition polymorphonuclear leukocyte-enriched preparations (fraction P, table 1) showed the highest aptitude to bind labeled TSH. It was therefore possible that TSH binding was related to the presence of the phagocytic cells, monocytes and granulocytes. Fig. 2 is a compilation of experiments in which TSH binding and the percent of phagocytic cells were determined. A significant correlation between both parameters was observed in 10 experiments. That [ 1251]TSH was reversibly bound to superficial cell sites and not ingested by the cells was ascertained by the following observations: (1) release of [1251] TSH in the presence or absence of excess unlabeled TSH was a rapid phenomenon, 50% of the bound hormone being released in 15 min (fig. 3); (2) control experiments in which [ ‘251]TSH was replaced by rat [125 Ilcarbonic anhydrase C showed the absence of specific binding to all the preparations of leukocytes studied; (3) preincubation (15 min) in the presence of latex beads inhibited by 80% the binding of [1251]TSH which would not have occurred if the cells had ingested the labeled hormone; (4) iodoacetamide, which fully inhibited endocytosis at a concentration of 0.1 mM, failed to prevent binding of TSH, whereas Ca*+ ions, which enhance phagocytosis, almost fully inhibited [ ‘25 I] TSH binding (table 2). It is also worth mentioning that unlabeled TSH at a concentration of 1 PM inhibited phagocytosis by 20%.
* 0.003 d - 0.074) + 0.04 0.26)
97.6 f 0.3 (97 - 99) 84.3 + 2.5 (75 -92)
86.1 i 2.3 (70 -95) 7.0 f 1.1 (4-12)
Lymphocytes (%)
b
2.1 f 0.3 (0.8 - 3) 12.8 * 3.0 (4-25)
10.6 f 1.6 (4 - 20) 3.0 f 0.7 (l-5)
Monocytes b (%)
0.36 f 0.16 (O-1) 0.85 f 0.30 (0.8 - 1.4)
1.1 t 0.1 (0.15-2) 88.6 f 1.4 (84-95)
Polymorphonuclear leukocytes b (%)
4.9 f 1.8 (0 - 14) 22.0 + 4.8 (7-33)
18.0 +-2.1 (7 - 29) 71.4 i 4.6 (50-86)
Phagocytic cells ’ (%)
staining; ’ Phagocytosis of Latex beads; d mean percentages F SEM, range in parentheses.
0.022 + 0.009 (0 - 0.043) 0.070 f 0.016 (0.014 - 0.097)
0.033 (0.014 0.22 (0.02 -
Specific binding a (%)
a To 1 X lo6 cells; b May-Griinewald-Giemsa
Adherent cells (fraction M)
Sephadex G-10 columns Nonadherent cells
Ficoll-Angiocontrix gradients Cells equilibrating at d = 1.077 (Fraction L) Cells equilibrating at d = 1.20 (Fraction P)
Cell population
Table 1 Binding of [ *251]TSH to leukocytes.
fi. 0 Q 2
CT f B D El s
g.
84
%
PHA~OCYTIC
CELLS
Fig. 2. Specific [I 251] TSH binding as a function of the amount of phagocytic cells in different cell fractions: 0, lymphocyte (fraction L); o, Sephadex G-10 nonadherent cells; 0, Sephadex G10 adherent cells (fraction M); 4, polymorphonuclear leukocytes (fraction P); 5-25 x lo6 cells/ml were incubated at 24°C with 0.9 nM [ 1251]TSH.
characteris tics of TSH receptor sites
In all the preparations, [1251~TSH-sp~ci~c binding was linearly related to cell concentration up to saturation. Fig. 4 represents a typical Scatchard plot of saturation experiments using a preparation of mononuclear lymphocytes. The plot is curvilinear with an upward concavity suggesting the presence of two types of binding sites with equilibrium association constants of 2.0 nM_’ and 0.3 @i-l. The same experiments using polymorphonuclear-enriched preparations gave K, of 1
t 10
I
I
30 MINUTES
I
I
I
_
50
Fig. 3. Dissociation of [ 12sI]TSH from leukocytes. Cells (15-20 X lo6 cells/ml) were preincubated with 1.1 nM [ 1251]TSH for 45 min at 24°C. After centrifugation and washing, the cells were post-incubated in the saine medium with (0) or without (0) unlabeled TSH (1 PM).
I1 25~]T~yro~ropin binding to leukocytes
85
Table 2 Some factors affecting [ 1251]TSH binding to leukocytes. Leukocytes (Fraction L) were incubated as described in Methods. Addition
Specific binding (%)
None Latex beads a (preincubation) Sodium aside (3 mM) Iodoacetamide (0.1 mM) CaCl2 (1.7 mM)
100 20 0 100 3
a 3.7
X
lo8 latex beads; 15 min preincubation.
r&l-’ and 4.3 @vI-‘. If it is accepted that only monocytes or polymorphonuclear leukocytes bind TSH and taking into account the high affinity sites, the number of sites per cell was estimated as 230 for monocytes and 140 for polymorphonuclear leukocytes.
DISCUSSION The aim of this study was to identify in human peripheral blood the white cells binding TSH. Fractionation of leukocytes by Ficoll-Angiocontrix gradients and Sephadex G-10 adherence columns led to preparations enriched in mononuclear and polymorphonuclear leukocytes of good viability (about 95%). Specific TSH receptor sites were detected in all the fractions studied. However, a clear correlation was found between TSH binding and the percentage of phagocytic cells (monocytes and polymorphonuciear leukocytes) present in the preparations,
I
I
I
0.2
i&ND ( ng/ml)
Fig. 4. Plot of bound/free TSH as a function of bound TSH to leukocytes (fraction L). 20-25 x lo6 cells/ml were incubated with 0.9 nM [ 1251]TSH and increasing amounts of unlabeled TSH. The same experiment performed with increasing amounts of [ 1251]TSH gave identical results.
86
0. Chabaud, S. Lissitzky
suggesting as already shown for insulin-binding (Schwartz et al., 1975) that lymphocytes are not able to bind TSH. Binding of [ 125I] TSH to monocytes and polymorphonuclear leukocytes is reversible and saturable. Both cells exhibit high-affinity, low-capacity and low-aftinity, highcapacity sites. However, it is not known whether these sites are present in each TSH-bind~g cell or whether there exist two populations of cells with receptors of different affinities for the hormone. Moreover, the curvilinear Scatchard plots of B/F versus B can also be interpreted by negative cooperativity of binding sites (De Meyts et al., 1973; 1976) or by aggregation of the ligand (Cuatrecasas and Hollenberg, 1975). The high-affinity receptor site (K, about 1 nM_‘) of phagocytic leukocytes is similar to that found in isolated porcine thyroid cells and their derived membranes (Verrier et al., 1974; Lissitzky et al., 1975). With this material no low-affinity receptor sites were detected. However, two types of sites of different affinities were shown in thyroid plasma membranes purified from bovine thyroid glands (Amir et al., 1973;Moore and Wolff, 1974). The possibility that TSH was ingested by cells by phagocytosis was ruled out by several types of controls: (1) 0.1 M iodoacetamide, which fully inhibits endocytosis of latex beads, was without effect on TSH binding; (2) Ca2+, which enhances phagocytes, inhibited TSH binding. However, in contrast to insulin binding to monocytes, sodium azide (3 mM) fully inhibited TSH binding (table 2). On the other hand, phagocytosis of latex beads partially inhibited TSH binding. Incubation of leukocytes in the presence both of latex beads and [ r251] TSH moderately inhibited TSH binding (22%) whereas preincubation of cells with latex beads prior to addition of [ 1251]TSH resulted in 80% binding inhibition. These results suggest that a direct relation exists between TSH binding and the phagocytic state of the cell, phagocytosis perhaps resulting in the masking of TSH-binding sites. The observation that preincubation in the presence of an excess of unlabeled TSH decreased but did not abolish phagocytosis suggests that TSH restricts the phagocytic capacity of monocytes and polyl~orphonuclear leukocytes. The interpretation of these results is not unambiguous. What could be the mechanism of action of TSH on the phagocytosis process in leukocytes? Stoic (1972a,b) showed that TSH in polymorphonuclear leukocytes decreased iodide uptake and organification, and phagocytosis, but increased cyclic AMP level. Also, prostaglandin E, increased cyclic AMP levels (Stoic, 1974) which in turn reduced adhesion of cells to glass capillary tubes (Bryant and Sutcliffe, 1974). Therefore, it seems that inhibition of phagocytosis could be regulated by intracellular cyclic AMP. Our findings that phagocytic leukocytes contain TSHspecific receptor sites strengthens the idea that this hormone may play a role in the regulation of phagocytosis by leukocytes.
[‘251]Thyrotropin
binding to leukocytes
81
ACKNOWLEDGEMENTS We are much indebted to Dr P. Mercier (Centre de Transfusion, Marseille) for helpful suggestions in the preparation of leukocyte fractions, to the Centre de Transfusion (Marseille) for the generous gift of human peripheral blood, to Dr. P. Jaquet and to B. Verrier for the 1251-labeled TSH preparations and to N. Limozin for the ‘251-labeled carbonic anhydrase preparations.
REFERENCES Amir, S.M., Carraway, T.F., Jr., Kohn, L.D. and Winand, R. (1973) J. Biol. Chem. 248,40924099. Archer, J.A., Gorden, P., Gavin, J.R., III, Lesniak, M.A. and Roth, J. (1973) J. Clin. Endocrinol. Metab. 36, 627-633. Bryant, R.E. and Sutcliffe, M.C. (1974) J. Clin. Invest. 54, 1241-1244. Cline, M.J. and Lehrer, R.I. (1968) Blood 32,423-435. Cuatrecasas, P. and Hollenberg, M.D. (1975) Biochem. Biophys. Res. Commun. 62, 31-41. De Meyts, P., Roth, J., Neville, D.M., Jr., Gavin, J.R., III, and Lesniak, M. (1973) Biochem. Biophys. Res. Commun. 55, 154-161. De Meyts, P., Raffaele Bianco, A. and Roth, J. (1976) J. Biol. Chem. 251, 1877-1888. English, D. and Andersen, B.K. (1974) J. Immunol. Meth. 5, 249-252. Jaquet, P., Hennen, G. and Lissitzky, S. (1974) Biochimie 56, 769-774. Lissitzky, S., Fayet, G., Verrier, B., Hennen, G. and Jaquet, P. (1973) FEBS Lett. 29, 20-24. Lissitzky, S., Fayet, G. and Verrier, B. (1975) Adv. Cyclic Nucleotide Res. 5, 133-152. Ly, I.A. and Mishell, R.I. (1974) J. Immunol. Meth. 5, 239-247. Moore, W.V. and Wolff, J. (1974) J. Biol. Chem. 249,6255-6263. Mullin, B.R., Lee, G. Ledley, F.D., Winand, R.J. and Kohn, L.D. (1976) Biochem. Biophys. Res. Commun. 69, 55-62. Schwartz, R.H., Bianco, A.R., Handwerger, B.S. and Kahn, CR. (1975) Proc. Natl. Acad. Sci. USA, 72,474-478. Stoic, V. (1971) Biochim. Biophys. Res. Commun. 45, 159-166. Stoic, V. (1972a) Endocrinology 91, 835-839. Stoic, V. (1972b) Biochim. Biophys. Acta 264, 285-288. Stoic, V. (1974) Blood 43,743-748. Verrier, B., Fayet, G. and Lissitzky, S. (1974) Eur. J. Biochem. 42, 355-365. White, J.E. and Engel, F.L. (1958) J. Clin. Invest. 37, 1556. Wolff, J. and Jones, A.B. (1971) J. Biol. Chem. 246,3939-3947. Yamashita, K. and Field, J.B. (1970) Biochem. Biophys. Res. Commun. 40, 171-178.
THYROTROPIN-SPECIFIC BINDING TO HUMAN PERIPHERAL MONOCYTES AND POLYMORPHONUCLEAR LEUKOCYTES
BLOOD
Odile CHABAUD and Serge LISSITZKY Laboratoire de Biochimie M6dicale et U38 de 1‘INSERM, Facultt de Mdecine, Moulin, 13385 Marseille Cedex 4, France
Received 19 July 1916;accepted
27 Bd Jean-
14 September 1916
[ *251]Labeled thyrotropin binding to leukocytes has been studied using lymphocyte-, monocyte- and polymorphonuclear leukocyte-enriched preparations obtained by centrifugation on Ficoh-Angiocontrix gradients and Sephadex G-10 adherence. From the relation between thyrotropin binding and phagocytosis as shown by latex beads ingestion, it is concluded that the hormone binds essentially to monocytes and polymorphonuclear leukocytes. Equilibrium association constants of the high-affinity, low-capacity sites (1 nM_l) are similar to those found in isolated thyroid cells or in thyroid plasma membranes. The role of thyrotropin in the regulation of phagocytosis by leukocytes is discussed. Keywords:
thyrotropin;
monocytes; polymorphonuclear
leukocytes; phagocytosis.
It is well established that thyrotropin (TSH), the physiological thyroid stimulator, binds to specific receptor sites located at the external surface of the plasma membrane of the follicular thyroid cell. In addition to its action on the thyroid cell, thyrotropin can also modulate some activities in other tissues. The hormone increases lipolysis in rat and guinea pig adipose tissue (White and Engel, 1958) and recently thyrotropin receptor sites were described in purified plasma membranes from human and guinea pig adipocytes (Mullin et al., 1976). Also, thyrotropin or an associated pituitary factor was shown to decrease iodide metabolism in human polymorphonuclear leukocytes (Stoic, 1972). In the thyroid, the earliest effect of thyrotropin is the activation of adenylate cyclase (Pastan and Katzen, 1967; Yamashita and Field, 1970; Wolff and Jones, 1971; Verrier et al., 1974) a plasma membrane enzyme. In polymorphonuclear leukocytes, an increase in intracellular cyclic AMP was observed after addition of thyrotropin or prostaglandin Et (Stoic, 1972, 1974) suggesting that gra?ulocytes may contain receptor sites specific to thyrotropin. In the present communication, we describe the presence of thyrotropin-specific receptor sites in human leukocytes and especially in monocytes and polymorphonuclear leukocytes. 19
80
MATERIAL
0. Chabaud, S. Lissitzky
AND METHODS
Materials Sephadex G-10 and Ficoll were obtained from Pharmacia (Uppsala, Sweden) and Angiocontrix 48 from Laboratories Guerbet (Aulnay-sous-Bois, France). Purified porcine thyrotropin (20 U/mg in the McKenzie bioassay in the mouse) was kindly donated by Dr G. Hennen (LiBge). CeZZpreparations All the media and glasswares were sterilized before use. Human peripheral blood (100-l 50 ml) obtained from normal volunteers was collected on heparin. After dilution (1 : 1) with phosphate-buffered saline, pH 7.2, fractionation was performed on Ficoll [6.2% (w/v)] -Angiocontrix [ 11.4? (v/v)] gradients (d = 1.077 g/ml) according to the method of English and Andersen (1974). Centrifugation was for 20 min at 400g at the barrier in a swinging rotor. The interfaces were collected, pooled and the cells were washed twice and counted. Polymorphonuclear leukocytes were obtained using an additional layer of Ficoll-Angiocontrix (d = 1 .I2 g/ml). Viability as shown by exclusion of erythrosin B (0.1%) was on the average about 95%. Cells were suspensed in the appropriate assay medium and were stored at 0°C until utilization. Cell identification Lymphocytes, monocytes and polymorphonuclear leukocytes were identified by morphological criteria after staining with May-GriinewaldGiemsa (MGG) stain. Monocytes and polymorphonuclear leukocytes were also characterized by their aptitude to ingest latex beads (0.8 pm; 7.4 X lo8 per assay). Assays were performed for 30-45 min at 37°C in the medium used for [l*‘I]TSH binding experiments without EDTA and in the presence of 20% calf serum according to Cline and Lehrer (1968). Phagocytosis was monitored by microscopic examination of leukocyte smears stained by MGG or directly under phase-contrast microscopy. This was done by retention on SephaSeparation of lymphocytes and monocytes dex G-10 as proposed by Ly and Mishell (1974) for the separation of spleen cells. Monocyte-enriched preparations were obtained from the Sephadex G-10 columns after removal of the nonadherent cells and vortexing the Sephadex in excess medium as indicated by Schwartz et al: (1975). The yield in nonadherent lymphocytes was 56% of the cells put on the column and that in monocytes detached from the gel about 15%. Binding of (l*‘I/ TSH Porcine TSH was labeled with “‘1 using the lactoperoxidase method of iodide oxidation to a specific radioactivity of 60 @i/pg (Jaquet et al., 1974). Binding assays were performed in 25 mM Tris-Cl, 120 mM NaCl, 1.2 mM MgS04, 5 mM KCl, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, pH 7.4, containing 1 mg/ml bovine serum albumin (Archer et al., 1973). Cells were
[i25i]Thyrotropin
binding to leukocytes
81
suspended in the binding medium at a concentration of 15-50 X 1O6 cells/ml after isolation from Ficofl-Angiocontrix gradients and at a concentration of 4-15 X IO6 cells/ml after separation on Sephadex G-l 0. Binding assays were performed in I X 7 cm polyethylene tubes in 100 or 200 d final voiume at 24’C using 20-25 ng [lz51] TSH per ml. After the appropriate time of incubation, 4 ml of cold medium was added, the cells were separated by centrifugation as previously described (Lissitzky et al., 1973) and cell-associated radioactivity was counted. Specific binding was obtained by subtracting from the total radioactivity bound the amount which was not displaced by an excess of unlabeled TSH (1 PM). The experimental points in figures correspond to closely agreeing duplicates. Typical experiments are shown.
RESULTS Ceil preparations In 25 experiments, the average yield of cells obtained by Ficoll-Angiocontrix gradients (c! = 1.077 g/ml) (fraction L) was 1.2 f 0.074 X lo6 cells/ml blood. This fraction contained 86.1 + 2.3% lymphocytes, 10.6 + 1.6% monocytes and 1.1 + 0.1% polymorphonculear leukocytes. As shown by latex particle ingestion, the number of phagocytic cells was 18.0 + 2.1%. Platelets were in very low amount and red blood cells practically absent. After filtration on Sepahdex G-10, nonadherent cells contained 2.1 f 0.3% (MGG determination) and 4.9 + 1.8% (latex bead ingestion) monocytes. Adherent cehs (fraction M) released by vortexing of the Sephadex contained 12.8 + 3.0% (MGG determination) and 22.0 + 4.8% (latex bead ingestion) monocytes. Agreement between the two methods of monocyte estimation is not very good. Since identification by morphological criteria is somewhat difficult, characterization by phagocytic properties is likely to be quantitatively more reliable, contamination by polymorphonuclear leukocytes being less than 1%. The fractions enriched in polymorphonuclear leukocytes (fraction P) contained about 88% of these cells of which 71% were able to ingest latex beads. Contamination by monocytes was about 4% and varied for Iymphocytes between 4 and 10%. Binding of [ ’ 2 51Jthyro tropin fraction Binding of [ 12’ I] TSH was initially assayed on the lymphocyte-enriched L. The time-course of hormone binding (fig. 1) showed that equilibrium between association and dissociation was obtained in about 4.5 min. In 13 experiments the average specific TSH binding was 0.033 + 0.003% of the 0.9 nM [12’1] TSH added to the cells. This very low binding capacity suggested that all the cells present in the preparation were not able to bind the hormone. On the basis of the observations of Stoic (1971, 1972a), on the inhibitory effect of TSH on iodine metabolism in polymorphonuclear leukocytes and those of Schwartz et al. (1975) on the bind-
82
Fig. 1. Time-course
0. Chabaud, S. Lissitzky
of specific
CYtes,2 experiments); 25-40
[ ‘25I] TSH binding to leukocytes (fraction L: 4 and 6% monoX lo6 cells/ml were incubated at 24°C in the presence of 0.9 nm
[‘251]TSH.
ing of insulin to monocytes, it is possible that these cells might be responsible for TSH binding. This was ascertained using monocyte-enriched preparations of mononuclear leukocytes. Table 1 shows that when the percentage of monocytes as shown by their phagocytic property was increased from about 4% to about 20% by passage over Sephadex G-10 columns, a 3-fold increase in TSH specific binding was observed. In addition polymorphonuclear leukocyte-enriched preparations (fraction P, table 1) showed the highest aptitude to bind labeled TSH. It was therefore possible that TSH binding was related to the presence of the phagocytic cells, monocytes and granulocytes. Fig. 2 is a compilation of experiments in which TSH binding and the percent of phagocytic cells were determined. A significant correlation between both parameters was observed in 10 experiments. That [ 1251]TSH was reversibly bound to superficial cell sites and not ingested by the cells was ascertained by the following observations: (1) release of [1251] TSH in the presence or absence of excess unlabeled TSH was a rapid phenomenon, 50% of the bound hormone being released in 15 min (fig. 3); (2) control experiments in which [ ‘251]TSH was replaced by rat [125 Ilcarbonic anhydrase C showed the absence of specific binding to all the preparations of leukocytes studied; (3) preincubation (15 min) in the presence of latex beads inhibited by 80% the binding of [1251]TSH which would not have occurred if the cells had ingested the labeled hormone; (4) iodoacetamide, which fully inhibited endocytosis at a concentration of 0.1 mM, failed to prevent binding of TSH, whereas Ca*+ ions, which enhance phagocytosis, almost fully inhibited [ ‘25 I] TSH binding (table 2). It is also worth mentioning that unlabeled TSH at a concentration of 1 PM inhibited phagocytosis by 20%.
* 0.003 d - 0.074) + 0.04 0.26)
97.6 f 0.3 (97 - 99) 84.3 + 2.5 (75 -92)
86.1 i 2.3 (70 -95) 7.0 f 1.1 (4-12)
Lymphocytes (%)
b
2.1 f 0.3 (0.8 - 3) 12.8 * 3.0 (4-25)
10.6 f 1.6 (4 - 20) 3.0 f 0.7 (l-5)
Monocytes b (%)
0.36 f 0.16 (O-1) 0.85 f 0.30 (0.8 - 1.4)
1.1 t 0.1 (0.15-2) 88.6 f 1.4 (84-95)
Polymorphonuclear leukocytes b (%)
4.9 f 1.8 (0 - 14) 22.0 + 4.8 (7-33)
18.0 +-2.1 (7 - 29) 71.4 i 4.6 (50-86)
Phagocytic cells ’ (%)
staining; ’ Phagocytosis of Latex beads; d mean percentages F SEM, range in parentheses.
0.022 + 0.009 (0 - 0.043) 0.070 f 0.016 (0.014 - 0.097)
0.033 (0.014 0.22 (0.02 -
Specific binding a (%)
a To 1 X lo6 cells; b May-Griinewald-Giemsa
Adherent cells (fraction M)
Sephadex G-10 columns Nonadherent cells
Ficoll-Angiocontrix gradients Cells equilibrating at d = 1.077 (Fraction L) Cells equilibrating at d = 1.20 (Fraction P)
Cell population
Table 1 Binding of [ *251]TSH to leukocytes.
fi. 0 Q 2
CT f B D El s
g.
84
%
PHA~OCYTIC
CELLS
Fig. 2. Specific [I 251] TSH binding as a function of the amount of phagocytic cells in different cell fractions: 0, lymphocyte (fraction L); o, Sephadex G-10 nonadherent cells; 0, Sephadex G10 adherent cells (fraction M); 4, polymorphonuclear leukocytes (fraction P); 5-25 x lo6 cells/ml were incubated at 24°C with 0.9 nM [ 1251]TSH.
characteris tics of TSH receptor sites
In all the preparations, [1251~TSH-sp~ci~c binding was linearly related to cell concentration up to saturation. Fig. 4 represents a typical Scatchard plot of saturation experiments using a preparation of mononuclear lymphocytes. The plot is curvilinear with an upward concavity suggesting the presence of two types of binding sites with equilibrium association constants of 2.0 nM_’ and 0.3 @i-l. The same experiments using polymorphonuclear-enriched preparations gave K, of 1
t 10
I
I
30 MINUTES
I
I
I
_
50
Fig. 3. Dissociation of [ 12sI]TSH from leukocytes. Cells (15-20 X lo6 cells/ml) were preincubated with 1.1 nM [ 1251]TSH for 45 min at 24°C. After centrifugation and washing, the cells were post-incubated in the saine medium with (0) or without (0) unlabeled TSH (1 PM).
I1 25~]T~yro~ropin binding to leukocytes
85
Table 2 Some factors affecting [ 1251]TSH binding to leukocytes. Leukocytes (Fraction L) were incubated as described in Methods. Addition
Specific binding (%)
None Latex beads a (preincubation) Sodium aside (3 mM) Iodoacetamide (0.1 mM) CaCl2 (1.7 mM)
100 20 0 100 3
a 3.7
X
lo8 latex beads; 15 min preincubation.
r&l-’ and 4.3 @vI-‘. If it is accepted that only monocytes or polymorphonuclear leukocytes bind TSH and taking into account the high affinity sites, the number of sites per cell was estimated as 230 for monocytes and 140 for polymorphonuclear leukocytes.
DISCUSSION The aim of this study was to identify in human peripheral blood the white cells binding TSH. Fractionation of leukocytes by Ficoll-Angiocontrix gradients and Sephadex G-10 adherence columns led to preparations enriched in mononuclear and polymorphonuclear leukocytes of good viability (about 95%). Specific TSH receptor sites were detected in all the fractions studied. However, a clear correlation was found between TSH binding and the percentage of phagocytic cells (monocytes and polymorphonuciear leukocytes) present in the preparations,
I
I
I
0.2
i&ND ( ng/ml)
Fig. 4. Plot of bound/free TSH as a function of bound TSH to leukocytes (fraction L). 20-25 x lo6 cells/ml were incubated with 0.9 nM [ 1251]TSH and increasing amounts of unlabeled TSH. The same experiment performed with increasing amounts of [ 1251]TSH gave identical results.
86
0. Chabaud, S. Lissitzky
suggesting as already shown for insulin-binding (Schwartz et al., 1975) that lymphocytes are not able to bind TSH. Binding of [ 125I] TSH to monocytes and polymorphonuclear leukocytes is reversible and saturable. Both cells exhibit high-affinity, low-capacity and low-aftinity, highcapacity sites. However, it is not known whether these sites are present in each TSH-bind~g cell or whether there exist two populations of cells with receptors of different affinities for the hormone. Moreover, the curvilinear Scatchard plots of B/F versus B can also be interpreted by negative cooperativity of binding sites (De Meyts et al., 1973; 1976) or by aggregation of the ligand (Cuatrecasas and Hollenberg, 1975). The high-affinity receptor site (K, about 1 nM_‘) of phagocytic leukocytes is similar to that found in isolated porcine thyroid cells and their derived membranes (Verrier et al., 1974; Lissitzky et al., 1975). With this material no low-affinity receptor sites were detected. However, two types of sites of different affinities were shown in thyroid plasma membranes purified from bovine thyroid glands (Amir et al., 1973;Moore and Wolff, 1974). The possibility that TSH was ingested by cells by phagocytosis was ruled out by several types of controls: (1) 0.1 M iodoacetamide, which fully inhibits endocytosis of latex beads, was without effect on TSH binding; (2) Ca2+, which enhances phagocytes, inhibited TSH binding. However, in contrast to insulin binding to monocytes, sodium azide (3 mM) fully inhibited TSH binding (table 2). On the other hand, phagocytosis of latex beads partially inhibited TSH binding. Incubation of leukocytes in the presence both of latex beads and [ r251] TSH moderately inhibited TSH binding (22%) whereas preincubation of cells with latex beads prior to addition of [ 1251]TSH resulted in 80% binding inhibition. These results suggest that a direct relation exists between TSH binding and the phagocytic state of the cell, phagocytosis perhaps resulting in the masking of TSH-binding sites. The observation that preincubation in the presence of an excess of unlabeled TSH decreased but did not abolish phagocytosis suggests that TSH restricts the phagocytic capacity of monocytes and polyl~orphonuclear leukocytes. The interpretation of these results is not unambiguous. What could be the mechanism of action of TSH on the phagocytosis process in leukocytes? Stoic (1972a,b) showed that TSH in polymorphonuclear leukocytes decreased iodide uptake and organification, and phagocytosis, but increased cyclic AMP level. Also, prostaglandin E, increased cyclic AMP levels (Stoic, 1974) which in turn reduced adhesion of cells to glass capillary tubes (Bryant and Sutcliffe, 1974). Therefore, it seems that inhibition of phagocytosis could be regulated by intracellular cyclic AMP. Our findings that phagocytic leukocytes contain TSHspecific receptor sites strengthens the idea that this hormone may play a role in the regulation of phagocytosis by leukocytes.
[‘251]Thyrotropin
binding to leukocytes
81
ACKNOWLEDGEMENTS We are much indebted to Dr P. Mercier (Centre de Transfusion, Marseille) for helpful suggestions in the preparation of leukocyte fractions, to the Centre de Transfusion (Marseille) for the generous gift of human peripheral blood, to Dr. P. Jaquet and to B. Verrier for the 1251-labeled TSH preparations and to N. Limozin for the ‘251-labeled carbonic anhydrase preparations.
REFERENCES Amir, S.M., Carraway, T.F., Jr., Kohn, L.D. and Winand, R. (1973) J. Biol. Chem. 248,40924099. Archer, J.A., Gorden, P., Gavin, J.R., III, Lesniak, M.A. and Roth, J. (1973) J. Clin. Endocrinol. Metab. 36, 627-633. Bryant, R.E. and Sutcliffe, M.C. (1974) J. Clin. Invest. 54, 1241-1244. Cline, M.J. and Lehrer, R.I. (1968) Blood 32,423-435. Cuatrecasas, P. and Hollenberg, M.D. (1975) Biochem. Biophys. Res. Commun. 62, 31-41. De Meyts, P., Roth, J., Neville, D.M., Jr., Gavin, J.R., III, and Lesniak, M. (1973) Biochem. Biophys. Res. Commun. 55, 154-161. De Meyts, P., Raffaele Bianco, A. and Roth, J. (1976) J. Biol. Chem. 251, 1877-1888. English, D. and Andersen, B.K. (1974) J. Immunol. Meth. 5, 249-252. Jaquet, P., Hennen, G. and Lissitzky, S. (1974) Biochimie 56, 769-774. Lissitzky, S., Fayet, G., Verrier, B., Hennen, G. and Jaquet, P. (1973) FEBS Lett. 29, 20-24. Lissitzky, S., Fayet, G. and Verrier, B. (1975) Adv. Cyclic Nucleotide Res. 5, 133-152. Ly, I.A. and Mishell, R.I. (1974) J. Immunol. Meth. 5, 239-247. Moore, W.V. and Wolff, J. (1974) J. Biol. Chem. 249,6255-6263. Mullin, B.R., Lee, G. Ledley, F.D., Winand, R.J. and Kohn, L.D. (1976) Biochem. Biophys. Res. Commun. 69, 55-62. Schwartz, R.H., Bianco, A.R., Handwerger, B.S. and Kahn, CR. (1975) Proc. Natl. Acad. Sci. USA, 72,474-478. Stoic, V. (1971) Biochim. Biophys. Res. Commun. 45, 159-166. Stoic, V. (1972a) Endocrinology 91, 835-839. Stoic, V. (1972b) Biochim. Biophys. Acta 264, 285-288. Stoic, V. (1974) Blood 43,743-748. Verrier, B., Fayet, G. and Lissitzky, S. (1974) Eur. J. Biochem. 42, 355-365. White, J.E. and Engel, F.L. (1958) J. Clin. Invest. 37, 1556. Wolff, J. and Jones, A.B. (1971) J. Biol. Chem. 246,3939-3947. Yamashita, K. and Field, J.B. (1970) Biochem. Biophys. Res. Commun. 40, 171-178.
