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Effects of insulin-like growth factors and growth hormone on the in vitro proliferation of T lymphocytes
Journal of Neuroimmunology, 38 (1992) 95-104
95
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02175
Effects of insulin-like growth factors and growth hormone on the in vitro proliferation of T lymphocytes R o n Kooijman
a, Mia Willems a, G e r T. Rijkers a, A d Brinkman c, Sylvia C. van Buul-Offers b, Cobi J. Heijnen a and Ben J.M. Zegers a
Department of Immunology, b Department of Endocrinology, UniversityHospitalfor Children and Youth "Het Wilhelmina Kinderziekenhuis", Utrecht, and c Department of Pathology I, Erasmus University, Rotterdam, The Netherlands (Received 4 November 1991) (Revised, received 23 December 1991) (Accepted 23 December 1991)
Key words: Insulin-like growth factor; Growth hormone; IGF-binding protein; Proliferation; T Lymphocyte; Mitogen
Summary The insulin-like growth factors I and II (IGF-I and IGF-II) promote proliferation and differentiation of many cell types. We report that recombinant IGF-I and IGF-II augment both the lectin- and anti-CD3-induced proliferation of human peripheral blood mononuclear ceils (PBMC) at concentrations proportional to their binding affinities. IGF-I and IGF-II also augmented the lectin-induced proliferation of purified T lymphocytes. Effects of IGF-I were found in cultures of T cells vigorously depleted for monocytes and supplemented with saturating concentrations of interleukin-1. The latter results indicate that the effect of IGF-I on the proliferation of T lymphocytes can occur independent of monocytes or monocyte-derived factors.
Introduction Growth hormone (GH) and the insulin-like growth factors (IGF-I and IGF-II) play a pivotal role in growth and development. The insulin-like growth factors comprise a family of peptides which promote proliferation and differentiation of various cell types (Froesch et al., 1985; Baxter, 1986). Although the distinction is certainly not absolute, it is assumed that IGF-II plays an im-
Correspondence to: R. Kooijman, Department of Immunology, Wilhelmina Kinderziekenhuis, P.O. Box 18009, 3501 CA Utrecht, The Netherlands.
portant role as a foetal growth factor, whereas IGF-I is more important for postnatal growth and development. Specific receptors for both growth factors have been described. The type I IGF receptor binds IGF-I and IGF-II with a high affinity, whereas the type II receptor binds IGF-II with a high affinity and IGF-I with a low affinity (Rechler and Nissley, 1985). It is generally accepted that the growth promoting effects of growth hormone, which become apparent after birth, are mediated by IGF-I (Isaksson et al., 1987). IGF-I is predominantly synthesized in the liver, but it can also be produced by a variety of tissues where it can act as an autocrine or paracrine growth factor (D'Ercole et al., 1984).
96 It is becoming evident that IGF-binding proteins may modulate many of the effects of IGFs in different tissues. Whereas most IGFs in the circulation are bound to a 150-kDa IGF-binding protein (IGFBP-3), the low molecular mass binding proteins are the main binding proteins in other body fluids. There is ample evidence that IGFBPs can modulate the growth promoting effects of IGFs. In various biological systems, IGFBPs have been shown to inhibit or potentiate IGF action (reviews by Hintz, 1990, Baxter, 1991). A role for IGF-I in the immune system is suggested by the expression of IGF-I in human macrophages and lymphoblastoid cell lines (Rom et al., 1988; Merimee et al., 1989), its ability to reverse thymic involution in diabetic rats (Binz et al., 1990), and its potent ability to activate neutrophils for superoxide anion production (Fu et al., 1991). Furthermore, both IGF-I and IGF-II might be involved in lymphocyte proliferation as suggested by the effects of both growth factors on the in vitro proliferation of lectin-activated lymphocytes (Schimpff et al., 1983; Tapson et al., 1988) and by the increased expression of type I and type II IGF receptors on phytohaemagglutinin (PHA) activated T cells (Thorsson and Hintz, 1977; Kozak et al., 1987; Tapson et al., 1988). Although IGF-I expression in a variety of cells and tissues can be regulated by factors other than GH, there are indications that GH can influence lymphocyte proliferation via secretion of IGF-I by lymphocytes. This is suggested by the observation that GH augments IGF-I secretion in Epstein Barr virus-transformed B cells (Merimee et al., 1989) and that the stimulatory effect of GH on the colony formation of Human T Cell Lymphoma Virus-I- and II-transformed T cells could be inhibited by antibodies against the type I receptor (Geffner et al., 1990). GH-induced IGF-I expression might play a pivotal role in the proposed interactions between GH and the immune system (Kelley, 1989). Results of in vitro studies on the effects of GH on the proliferation of peripheral blood mononuclear cells (PBMC) are contradictory, since both positive (Astaldi et al., 1973; Mercola et al., 1981) and negative (Kiess et al., 1983; Schimpff and Repellin, 1989) effects of GH have been reported. This may be due to the use of GH from different origins a n d / o r to the
culture conditions employed. Since the effects of GH may be mediated by IGF-I, it is important that experiments are performed under IGF-free conditions. The purpose of the present work was to determine whether IGF-I and IGF-II are involved in the in vitro proliferation of PBMC and in particularly of T cells. Since no dose-response relationship for IGFs using media without endogenous IGFs has been reported, we tested the effects of recombinant IGF-I and IGF-II on lymphocyte proliferation in serum-free medium and in fetal calf serum (FCS) that has been treated with dithiothreitol (DTT) in order to inactivate IGFs. We found that IGF-I and IGF-II augment the proliferation of PHA- and anti-CD3 activated PBMC and purified T cells at concentrations that are in accordance with their binding affinities.
Materials and methods
Cells
Human peripheral blood mononuclear cells (PBMC) were purified from heparinized blood from healthy adult donors by centrifugation on Ficoll/Isopaque (Pharmacia, Uppsala, Sweden) density gradients (1.077 g/ml) at 1000 x g for 20 min at room temperature. T cells were isolated from PBMC by rosetting with sheep erythrocytes that were pretreated with 2-aminoethyl-isothiouronium bromide (AET; Sigma, St. Louis, MO; Gmelig Meyling et al., 1977). This T cell fraction contained at least 90% CD3 positive cells and 1-2% monocytes as assessed by immunofluorescence cytometry using anti-CD3 (leu 4) and anti-CD14 (leu M3) as primary antibodies and fluorescein-conjugated goat-anti-mouse Ig as a secondary antibody (antibodies were obtained from Becton Dickinson, San Jose, CA). Residual monocytes in T cell-enriched preparations were removed by treatment with leucine methyl ester (Thiele et al., 1983). Chemicals and materials
Recombinant human growth hormone (Genotropin) was obtained from Kabivitrum (Stockholm, Sweden). Recombinant human IGF-I and IGF-II were kindly provided by Dr. Jeatran, Lilly
97 Research Laboratories, Indianapolis, IN. Human IGF-binding protein 1 (IGFBP-1) was purified from amniotic fluid (Drop et al., 1982). L-Leucine methyl ester and bovine serum albumin (BSA) were from Sigma (St. Louis, MO). The BSA did not contain any detectable IGF-activity as determined by radioimmunoassay. Recombinant IL-1/3 was a generous gift from Dr. A. Shaw, Glaxo Institute of Molecular Biology, Geneva, Switzerland. Normal human serum (NHS) was a pool from five normal adults and fetal calf serum (FCS) and was obtained from Gibco (Grand Island, NY). The FCS batches we used were selected for a low level of [3H]thymidine ([3H]dTd) incorporation by PBMC in the absence of mitogens (< 100 cpm). Growth factor-inactivated serum (SH-FCS) was prepared by treatment of FCS with 100 mM dithiothreitol (DTT; Sigma). After overnight dialyses against a 50-fold excess of phosphate-buffered saline, the formed sulfhydryl groups were alkylated with 5 mg/ml iodoacetamide (Sigma). DTT and iodoacetamide were removed by dialysis (molecular weight cutoff 8-10 kDa) against a 75-fold excess of phosphatebuffered saline which was repeated five times. A more detailed description of the procedure and further information about the composition of the inactivated serum has been presented by Van Zoelen et al. (1985). The anti-CD3 mAb RIV9 was a gift from Dr. J. Kreeftenberg, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands (Vaessen et al., 1989).
Cell culture and proliferation assays For PHA-induced proliferation, PBMC were cultured in round-bottom 96-well polysterene microtiter plates (Nunc, Kamstrup, Denmark) at a concentration of 2.7 X l0 s cells/ml in 150 /~1 medium containing RPMI 1640 (Gibco), 100 U / m l penicillin, 100/zg/ml streptomycin, 4 mM glutamine and 10% FCS (heat-inactivated: 45 min, 56°C) or 2% NHS (heat-inactivated). When the ceils were cultured in 10% SH-FCS or 0.2% BSA, the medium was supplemented with 12.5 /zg/ml transferrin and 30 nM sodium selenite. PBMC were stimulated with 15 /~g/ml phytohemagglutinin (PHA; HA15 Wellcome, Dartford, UK) and the T cells were activated with 40 /~g/ml PHA.
After 48 h of culture at 37°C in 5% C02, 100% relative humidity, the cells were pulsed with 37 kBq of [3H]dTd (1.65 TBq/mol; Amersham international, Amersham, UK) and then cultured for a further 18 h. When PBMC were activated with anti-CD3 (RIV9, 1:3000), they were cultured in flat-bottom wells at a concentration of 106 cells/ml (200 ~l total volume) for 72 h prior to the addition of [3H]dTd and were harvested 18 h thereafter onto glass fiber filters. The incorporated [3H]dTd was measured using standard scintillation procedures. The significance of observed differences between means were determined using Student's t-test.
Results
Effects of IGF-I and IGF-II on proliferation of human PBMC and T cells The effects of IGF-I and IGF-II on PHAactivated PBMC were tested using different culture conditions. When lymphocytes were cultured in 10% FCS, addition of GH, IGF-I or IGF-II did not influence the proliferation at concentrations that are consistent with their binding affinities for their receptors. Only high concentrations of IGF-I (i.e. 10 -7 M) increased proliferation. Since FCS contains high concentrations of IGF-I and IGF-II (Daughaday and Rotwein, 1989), we also tested the effects of growth factors using serum-free medium or medium supplemented with inactivated FCS (SH-FCS). The IGFs can be inactivated by reduction of disulfide bonds. As a result of inactivation with DTI', the IGF concentration in the serum decreases to less than 10 -11 M (Van Zoelen et al., 1985). Furthermore, at least certain IGF-binding proteins (IGFBPs) are inactivated by reduction (Hardouin et al., 1987), because the appropiate folding of the molecules depends on disulphide bonds (Brinkman et al., 1991). Replacement of FCS by SH-FCS supplemented with transferrin and sodium selenite resulted in a reduction of basal PHA-induced thymidine incorporation from 9500 to 5500 cpm (Fig. 1A). However, proliferation in this serum appeared to be 3 to 4 times higher than in a serum-free medium with bovine serum albumin (BSA) used at an equiva:lent protein concentration (data not pre-
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Fig. 1. Effects of recombinant human IGF-I (©), IGF-II (e) and G H (zx) on the proliferation of PBMC. A. PHA-activated cells were cultured for 66 h in 10% FCS (dashed lines) or 10% IGF-inactivated FCS (SH-FCS) supplemented with 12.5 / z g / m l transferrin and 30 nM sodium selenite (solid lines). B. Cells from the same donor were activated with an anti-CD3 mAb (RIV9) and cultured for 90 h in the medium with SH-FCS. Standard errors were below 10% of the mean. Asterisks indicate significant difference from control conditions (~ P < 0.05; * P < 0.005). These data are representative of four replicate experiments.
sented). The difference in cell proliferation between cultures performed in BSA or SH-FCScontaining medium could not be reduced by addition of extra transferrin (data not shown). Because of the higher proliferative response of the lymphocytes in SH-FCS, we preferred the use of this inactivated serum for most experiments. The PHA- and anti-CD3-induced proliferation of PBMC in 10% SH-FCS is augmented by IGF-I and IGF-II at concentrations that are in the order of magnitude of the free concentrations in serum and the K d of their receptors (Fig. 1A and B). The magnitude of the effects of IGF-I and IGF-II varied between individual donors, but the effects were always positive. For 10 -8 M IGF-I and IGF-II we found a mean increase in the proliferative response of 41% and 32%, respectively (Table 1). To test whether IGF-I and IGF-II can augment the proliferation of PBMC independently of other serum factors, we performed the same experiments in a serum-free medium which contains 0.2% BSA instead of SH-FCS. As depicted in Fig. 2, it appeared that IGF-I and
TABLE 1 MEAN EFFECTS OF IGF-I, IGF-II, G H A N D IGFBP-1 ON T H E P H A - I N D U C E D P R O L I F E R A T I O N O F PBMC Stimulation index + SD 10 9 M IGF-I 10 s M IGF-I 10 -9 M IGF-II 10 -8 M IGF-II 10 - s M GH 10 -7 M GH IGFBP-1 10 -8 M GH 10 -7 M GH
1.22+0.14 1.41 +0.27 1.15+0.16 1.32_+0.27 0.93+0.09 0.84_+0.08 0.70_+0.07 1.00+_0.04 0.94-+0.08
(n (n (n (n (n (n (n (n (n
= = = = = = = = =
7) 9) 5) 6) 6) 6) 6) 4) 4)
a
b b c J d
a The effects of IGF-I and IGF-II were measured in RPMI medium containing 10% SH-FCS. b Effects of G H in RPMI medium containing 10% SH-FCS. c Effects of IGF-binding protein 1 were studied on cells cultured in RPMI medium supplemented with 2% normal human serum (NHS). d Effects of G H measured in RPMI medium containing 0.2% BSA. The incorporation of [3H]dTd ranged between 3000 and 10000 cpm for cells cultured in RPMI containing NHS and between 1000 and 5000 cpm for the cells that were cultured in a medium with SH-FCS or BSA.
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[IGF] Fig. 2. Effects of recombinant human IGF-I ( o ) and IGF-II (e) on the proliferation of PHA-activated PBMC that were cultured for 72 h in RPMI containing 0.2% BSA, transferrin and selenite. The mean v a l u e s + S E M from quadruplicate wells are presented. These data are representative of three replicates.
IGF-II also augment the proliferation of PBMC in serum-free medium and that the dose-response curves for IGF-I and IGF-II were very similar to 40OO
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[hormone] Fig. 3. Effects of recombinant human IGF-I (o), IGF-II (e) and GH ( a ) on the proliferation of PHA-activated T lymphocytes. T cells were purified with AET-pretreated sheep erythrocytes and cultured in 10% SH-FCS supplemented with transferrin and sodium selenite in the presence of 4 0 / z g / m l P H A for 66 h in 96-well microtiter plates. Standard errors of the mean were below 5%. * Significantly different from control conditions (* P < 0.005). These data are representative of three replicate experiments.
the curves obtained using SH-FCS (see Fig. 1). The EDs0 of 0.12 nM for the effects of IGF-I in the serum-free system corresponds with the K d for the type I receptor on T cells (0.12 nM) as described by Tapson et al. (1988). The 10-fold higher IGF-II concentrations required to evoke the same effect as IGF-I reflect the affinity of IGF-II for the type I and type II receptors on lymphocytes (Kozak et al., 1987). In all of our experiments, the [3H]dTd incorporation was lower than 100 cpm when the cells were not treated with mitogen (data not presented). The effects of IGF-I and IGF-II on PHA-induced proliferation of purified T cells were much larger than those on PBMC (Fig. 3). IGF-I (10 nM) increased the proliferation of purified T cells obtained from different donors by 108 + 64% (n = 7) and IGF-II augmented proliferation by 78 + 21% (n = 3). The rosetted T cells, which could be in a partially activated state as a result of the rosetting procedure, did not respond to IGFs unless they were activated by PHA (data not shown). Although these results suggest that IGFs stimulate T cells directly, a role for monocytes in mediating the effects of IGFs cannot be excluded, because IGF-I receptor bearing monocytes (Stuart et al., 1991; manuscript in preparation) may contaminate the T cell fractions. To test whether IGF-I augments T cell proliferation via stimulation of monocyte-derived interleukin 1 (IL-1), we tested the effects of IGF-I on the proliferation of T cells in the presence of IL-1. It appeared that IGF-I still stimulated the proliferation of purified T cells in the presence of saturating levels IL-1 (Fig. 4A). These results indicate that the positive effect of IGF-I on T cell proliferation is not a secondary event due to an increased secretion of IL-1. To exclude other possible monocyte-mediated effects of IGF-I, the remaining monocytes in the T cell fraction were killed by incubation with leucine methyl ester. No monocytes could be detected in leucine methyl ester-treated T cells with a non-specific esterase staining (detection level 0.1%). As depicted in Fig. 4B, the effects of IGF-I on T cell proliferation were still observed in leucine methyl estertreated T cell preparations. When monocytes from the same donor, obtained by adherence onto plastic tissue flasks, were added to the cul-
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Fig. 4. Effects of IL-1 and monocytes on the augmentation of T cell proliferation by recombinant human IGF-I. A. Effects of IGF-I on PHA-induced T cell proliferation in the presence and absence of IL-1. All differences between the controls without IGF-I and the cultures with IGF-I are statistically significant (P < 0.05). Significant effects of IL-1 are indicated (11, P < 0.05). B. Effects of IGF-I on T cell proliferation in the absence and in the presence of different concentrations of monocytes. Open bars, control; stippled bars, 10 10 M IGF-I; cross-hatched bars, 10 - 9 M IGF-I; closed bars, 10 -s M IGF-I. Monocytes in the T cell fraction were killed by treatment with leucine methyl ester. Cell cultures with different concentrations monocytes were obtained by addition of monocytes that were isolated from PBMC by adherence onto plastic culture flasks. Statistically significant effects of IGF-I are indicated (* P < 0.05). The mean values + SEM from quadruplicate wells are presented. The data are representative of four experiments.
ture, we f o u n d t h a t th e positive effect of exogenous I G F - I on T cell p r o l i f e r a t i o n was r e d u c e d (Fig. 4B).
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Fig. 5. Inhibition of the proliferation promoting effect of recombinant IGF-I and IGF-II by IGF-binding protein 1 (IGFBP-1). PBMC were cultured for 66 h with 15 ~g/ml PHA in RPMI with 10% SH-FCS supplemented with transferrin and sodium selenite. The effects of different concentrations IGF-I and IGF-II were monitored in the presence (closed bars) and in the absence (open bars) of 5.4 nM IGFBP-1. The mean values _+SEM from quadruplicate wells are presented.
Effects of IGF-binding protein 1 In o r d e r to test t h e ability o f I G F B P - 1 to m o d u l a t e t h e effects o f I G F s on l y m p h o c y t e proliferation, we t e s t e d the effects of p u r i f i e d I G F B P - 1 o n the effects of I G F - I and I G F - I I on PHA-induced proliferation of PBMC. Figure 5 shows that t h e effects o f b o t h 0.4 n M r e c o m b i n a n t I G F - I and I G F - I I are a b r o g a t e d by 5.4 n M I G F B P - 1 . N o inhibitory effect o f I G F B P - 1 was f o u n d w h e n an excess o f I G F - I or I G F - I I was a d d e d . T o test t h e effects of I G F B P - 1 on t h e growth p r o m o t i n g effects o f native h u m a n I G F - I as p r e s e n t in serum, we c u l t u r e d P B M C in m e d i u m s u p p l e m e n t e d with 2 % n o r m a l h u m a n s e r u m ( N H S ) . It a p p e a r e d that a d d i t i o n o f 5.4 n M I G F B P - 1 i n h i b i t e d t h e m i t o g e n - i n d u c e d proliferation (Fig. 6). C o n c o m i t a n t a d d i t i o n o f I G F B P - 1 with excess I G F - I or I G F - I I ( 1 0 - 1 0 0 n M ) a b r o g a t e s t h e inhibitory effect of I G F B P - 1 (Fig. 6). T h e s e results show that the effects o f native an d r e c o m b i n a n t h u m a n I G F - I an d I G F - I I on t h e p r o l i f e r a t i o n o f P B M C can be i n h i b i t e d by I G F B P - 1 . A d d i t i o n of I G F B P - 1 to P B M C f r o m six d o n o r s c u l t u r e d in m e d i u m s u p p l e m e n t e d with 2 % N H S i n h i b i t e d the p r o l i f e r a t i o n of P H A act i v at ed l y m p h o cy t es f r o m 20 to 5 0 % with a
10l 15
BSA. Also lower concentrations (10 - u to 10 -9 M) were without any effect (data not presented). Similar to the results for IGF-I and IGF-II, GH was not able to induce proliferation of resting cells (data not presented).
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Fig. 6. Inhibition of lymphocyte proliferation by IGF-binding protein 1 (IGFBP-1). P B M C were cultured in R P M I medium with 2% normal h u m a n serum (NHS) and 15 ~ g / m l P H A for 72 h. The effect of the binding protein was tested in the absence or the presence of different concentrations recombinant IGF-I and IGF-II. The concentrations native IGF-I and IGF-II in 2% NHS are 0.4 nM and 1.6 nM respectively. Open bars, control; closed bars, with 5.4 nM IGFBP-1. The mean values + SEM from quadruplicate wells are presented.
mean of 30% for six donors (Table 1). This indicates that a substantial part of the growth-promoting effect of human serum on PBMC is caused by IGFs.
Effects of recombinant human growth hormone In order to be able to measure IGF-I-mediated effects of GH, and synergistic effects of GH with other serum factors, the effects of growth hormone were determined in either 10% SH-FCS or 10% FCS. With either serum, we did not find a positive effect of GH on thymidine incorporation when the cells were stimulated with either PHA or anti-CD3 (Fig. 1A, B). Indeed, the effects of GH on PHA-induced proliferation were slightly negative (Fig. 1 and Table 1). Even though the proliferation of purified T cells was strongly augmented by IGF-I, it was not influenced by GH (Fig. 3). To avoid the possible effects of bovine GH from FCS, which binds only weakly to human growth hormone receptors (Leung et al., 1987), we also tested the effects of human GH in serum free medium containing 0.2% BSA. As depicted in Table 1, high concentrations of GH had no effect on the proliferation of PBMC in 0.2%
We investigated the effects of IGF-I and IGFII on the proliferation of human lymphocytes in various culture media. IGF-I and IGF-II had no effect on the proliferation of PBMC cultured in 10% FCS (Fig. 1A) or 2% NHS (Fig. 6) at concentrations which are in accordance with their usual dissociation c o n s t a n t s ( 1 0 - 1 0 t o 10 - 9 M ) , while significant positive effects were observed when 0.2% BSA or 10% SH-FCS was used. When FCS was replaced by SH-FCS, the addition of IGF-I or IGF-II could not restore the proliferation to the level in FCS, indicating that besides IGFs, the DTT treatment has also inactivated other factors that promote lymphocyte proliferation. The higher level of thymidine incorporation in SH-FCS compared to the level in BSA may be due to the presence of DTl'-resistant growth factors or lipids (Van Zoelen et al., 1985) which are important for lymphocyte proliferation (Spieker-Polet and Polet, 1981; Cuthbert and Lipsky, 1986). The ED50 for IGF-I on proliferation of PBMC in 0.2% BSA and 10% SH-FCS is equivalent to the K d of 0.12 nM for the type I IGF receptor on resting and PHA-activated T lymphocytes (Tapson et al., 1988). The 10 times higher concentration of IGF-II that is necessary to induce the same effect as IGF-I is in accordance with the relatively lower binding affinity of this factor for the type I and type II receptor. Although there are indications that IGF-II effects can be mediated by the type II receptor (Tally et al., 1987), it is generally accepted that most growth-promoting effects of IGF-II take place via binding to the type I receptor (Nissley and Rechler, 1984; Zapf et al., 1984). The contribution of native IGFs in human serum to the proliferation of PBMC was assessed by addition of an excess of the binding protein to PBMC cultured in R P M I / 2 % NHS. The inhibitory effect was donor-dependent and varied from 22 to
102
51% with a mean of 30% (Table 1). Therefore, we conclude that the IGFs are a relatively important serum factor for the proliferation of PBMC. When IGF-I or IGF-II was added to purified T cells cultured in R P M I / 1 0 % SH-FCS, we found an even greater increase in proliferation as compared to the effects on PMBC. Since the effects of IGF-I were also observed in a monocyte-free T cell culture supplemented with a saturating concentration of IL-1, we conclude that IGF-I can enhance T cell proliferation in the absence of monocytes. The stimulation index of IGF-I appeared to be inversely related with the percentage monocytes in the culture. This effect may be the result of secretion of IGFs or IGF-binding proteins by monocytes, which is currently a subject of investigation. Both IGF-binding and IGF-I proteins can be produced by cells of the immune system (Rom et al., 1988; Merimee et al., 1989; Baxter et al., 1991; Neely et al., 1991). Although the effects of IGFs are in the range observed in many other cell systems, it is still possible that the effects of IGF-I on T cells is a relatively strong effect on only a fraction of the T cells. A functional role for IGFs in lymphocyte proliferation is suggested by their positive effects on proliferation and upregulation of their receptors (Kozak et al., 1987; Tapson et al., 1988). This idea is supported by the results of Hansson et al. (1988) who showed that cytoplasmic IGF-I is present in part of the lymphocytes in the thymus and the spleen of rats, and by the observation of Baxter et al. (1991) that rat leukocytes produce IGF-I. These results and our observation that PHA-activated human T cells contain immunoreactive IGF-I (unpublished results) indicate that IGF-I might have an autocrine or paracrine function in the immune system. G H has been implicated to interact with the immune system in experimental animals. GH-deficient mice such as the Snell-Bagg dwarf mice are immuno-deficient (Baroni, 1967; Duquesnoy, 1972; Van Buul-Offers, 1983) and hypophysectomized rats have an impaired cellular and humoral immune response which can be restored by treatment with G H (Kelley, 1989; Edwards et al., 1991). Also in vitro experiments with HTLVtransformed human cell lines demonstrate that G H augments T lymphocyte colony formation
(Geffner et al., 1990). In the latter study, it was demonstrated that the effects of G H were mediated by locally produced IGF-I. In addition, Baxter et al. (1991) showed that G H stimulates the production of IGF-I in rat leukocytes. Reports on the effects of human G H on the in vitro proliferation of peripheral lymphocytes have yielded conflicting results. To avoid effects of contaminating substances and to allow the growth-promoting effect of G H to be mediated by IGF-I, we studied the effects of recombinant human G H on lymphocyte proliferation in 10% SH-FCS and 0.2% BSA. Although it has been reported that human G H can induce blastogenesis (Mercola et al., 1981) and thymidine incorporation in unstimulated PBMC (Kiess et al., 1983), we did not find an effect of recombinant human G H on the thymidine incorporation of resting lymphocytes, not even when employing the culture conditions as described by Kiess et al. (1983; data not shown). The effects of G H on PHA- and anti-CD3-induced proliferation, if any, were negative. Negative effects were also found by Schimpff et al. (1989) and Kiess et al. (1983). Although we used serum-free culture conditions, our results do not exclude a possible role for G H in lymphocyte proliferation, because G H has been shown to be produced by isolated PBMC (Weigent and Blalock, 1991). Indeed, the inhibition of G H production in rat leukocytes inhibits the proliferation (Weigent et al., 1991). Overall, our results indicate that IGFs are important growth factors for proliferation of human T cells and that the effects of IGF-I can occur in the absence of monocytes. More insight in the role of IGFs in the immune system can be obtained by further investigations on the effects of IGFs and by studying the expression of IGFs and their receptors in cells of the immune system.
Acknowledgements We are indebted to Drs. K.W. Kelley and S. Arkins (University of Illinois, IL) for their critical assessment of this paper, to Dr. A. Shaw (Glaxo Institute for Molecular Biology, Geneva, Switzerland) for kindly providing riLl/3 and to Dr. Jeatran (Lilly Research Laboratories, Indianapo-
103
lis, IN) for providing recombinant IGF-I and IGF-II. Furthermore, we thank Dr. B. van den Burg for his advise for the preparation of IGF-inactivated serum. References Astaldi, A., Yalcin, A., Meardi, G., Burgio, G.R., Merolla, R. and Astaldi, G. (1973) Effects of growth hormone on lymphocyte transformation in cell culture. Blut 26, 74-81. Baroni, C. (1967) Thymus, peripheral lymphoid tissues, and immunological responsiveness of the pituitary dwarf mouse. Experientia 23, 282-283. Baxter, R.C. (1986) The somatomedins: insulin-like growth factors. Adv. Clin. Chem. 25, 49-115. Baxter, R.C. (1991) Physiological role of IGF binding proteins. In: E.M. Spencer (Ed.), Modern Concepts of Insulin-like Growth Factors. Elsevier, New York, NY, pp. 171-180. Baxter, J.B., Blalock, J.E. and Weigent, D.A. (1991) Characterization of immunoreactive insulin-like growth factor-I from leukocytes and its regulation by growth hormone. Endocrinology 129, 1727-1734. Binz, K., Joller, P., Froesch, P., Binz, H., Zapf, J. and Froesch, E.R. (1990) Repopulation of the atrophied thymus in diabetic rats by insulin-like growth factor I. Proc. Natl. Acad. Sci. USA 87, 3690-3694. Brinkman, A., Kortleve, D.J., Zwarthoff, E.C. and Drop, S.L.S. (1991) Mutations in the C-terminal part of IGF binding protein-I result in dimer formation and loss of IGF binding capacity. Mol. Endocrinol. 5, 987-994. Cuthbert, J.A. and Lipsky, P.E. (1986) Promotion of human T lymphocyte activation and proliferation by fatty acids in low density and high density lipoproteins. J. Biol. Chem. 261, 3620-3627. D'Ercole, A.J., Stiles, A.D. and Underwood, L.E. (1984) Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc. Natl. Acad. Sci. USA 81, 935-939. Daughaday, W.H. and Rotwein, P. (1989) Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. Rev. 10, 68-91. Drop, S.L.S., Kortleve, D. and Guyda, H. (1982) Isolation of somatomedin binding protein in human amniotic fluid; development of a radioimmunoassay. Pediatr. Res. 16, 890. Duquesnoy, R.J. (1972) Immunodeficiency of the thymus-dependent system of the Ames dwarf mouse. J. Immunol. 108, 1578-1590. Edwards, C.K., Yunger, L.M,, Lorence, R.M., Dantzer, R. and Kelley, K.W. (1991) The pituitary gland is required for protection against lethal effects of Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 88, 2274-2277.
Froesch, E.R., Schmid, C., Schwander, J. and Zapf, J. (1985) Actions of insulin-like growth factors. Annu. Rev. Physiol. 47, 443-467. Fu, Y.K., Arkins, S., Wang, B.S. and Kelley, K.W. (1991) A novel role of growth hormone and insulin-like growth factor-I. Priming neutrophils for superoxide anion secretion. J. Immunol. 146, 1602-1608. Geffner, M.E., Bersch, N., Lippe, B.M., Rosenfeld, R.G., Hintz, R.L. and Golde, D.W. (1990) Growth hormone mediates the growth of T-lymphoblast cell lines via locally generated insulin-like growth factor-I. J. Clin. Endocrinol. Metab. 71,464-469. Gmelig Meyling, F., Uytdehaag, A.C.G.M. and Ballieux, R.E. (1977) Human B activation in vitro. T dependent pokeweed mitogen-induced differentiation of blood B lymphocytes. Cell. Immunol. 33, 156-169. Hansson, H.A., Nilsson, A., Isgaard, J., Billig, H., Isaksson, O., Skottner, A., Andersson, I.K. and Rozell, R. (1988) Immunohistochemical localization of insulin-like growth factor I in the adult rat. Histochemistry 89, 403-410. Hardouin, S., Hossenlopp, P., Segovia, B., et al. (1987) Heterogeneity of insulin-like growth factor binding proteins and relationships between structure and affinity. 1. Circulating forms in man. Eur. J. Biochem. 170, 121-132. Hintz, R.L. (1990) Role of growth hormone and insulin-like growth-factor-binding proteins. Horm. Res. 33, 105-110. Isaksson, O.G., Lindahl, A., Nilsson, A. and Isgaard, J. (1987) Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr. Rev. 8, 426-438. Kelley, K.W. (1989) Growth hormone, lymphocytes and macrophages. Biochem. Pharmacol. 38, 705-713. Kiess, W., Holtmann, H., Butenandt, O. and Eife, R. (1983) Modulation of lymphoproliferation by human growth hormone. Eur. J. Pediatr. 140, 47-50. Kozak, R.W., Haskell, J.F., Greenstein, L.A., Rechler, M.M., Waldmann, T.A. and Nissley, S.P. (1987) Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes. Cell. Immunol. 109, 318-331. Leung, D.W., Spencer, S.A., Cachianes, G., et al. (1987) Growth hormone receptor and serum binding proteins: purification, cloning and expression. Nature 330, 537-543. Mercola, K.E., Cline, M.J. and Golde, D.W. (1981) Growth hormone stimulation of normal and leukemic human Tlymphocyte proliferation in vitro. Blood 58, 337-340. Merimee, T.J., Grant, M.B., Broder, C.M. and Cavalli Sforza, L.L. (1989) Insulin-like growth factor secretion by human B-lymphocytes: a comparison of cells from normal and pygmy subjects. J. Clin. Endocrinol. Metab. 69, 978-984. Neely, E.K., Smith, S.D. and Rosenfeld, R.G. (1991) Human leukemic T and B lymphoblasts produce insulin-like growth factor binding proteins 2 and 4. Acta Endocrinol. 124, 707-714. Nissley, S.P. and Rechler, M.M. (1984) Somatomedin/ insulin-like growth factor tissue receptors. Clin. Endocrinol. Metab. 13, 43-67. Rechler, M.M. and Nissley, S.P. (1985) The nature and regu-
104 lation of the receptors for insulin-like growth factors. Ann. Rev. Physiol. 47, 425-442. Rom, W.N., Basset, P., Fells, G.A., Nukiwa, T., Trapnell, B.C. and Crysal, R.G. (1988) Alveolar macrophages release an insulin-like growth factor I-type molecule. J. Clin. Invest. 82, 1685-1693. Schimpff, R.M. and Repellin, A.M. (1989) In vitro effect of human growth hormone on lymphocyte transformation and lymphocyte growth factors secretion. Acta Endocrinol. Copenh. 120, 745-752. Schimpff, R.M., Repellin, A.M., Salvatoni, A., Thieriot Prevost, G. and Chatelain, P. (1983) Effect of purified somatomedins on thymidine incorporation into lectinactivated human lymphocytes. Acta Endocrinol. Copenh. 102, 21-26. Spieker-Polet, H. and Polet, H. (1981) Requirement of a combination of a saturated and an unsaturated free fatty acid and a fatty acid carrier protein for in vitro growth of lymphocytes. J. Immunol. 126, 949-954. Stuart, C.A., Meehan, R.T., Neale, L.S., Cintron, N.M. and Furlanetto, R.W. (1991) Insulin-like growth factor-I binds selectively to human peripheral blood monocytes and Blymphocytes. J. Clin. Endocrinol. Metab. 72, 1117-1122. Tally, M., Li, C.H. and Hall, K. (1987) IGF-2 stimulated growth mediated by the somatomedin type 2 receptor. Biochem. Biophys. Res. Commun. 148, 811-816. Tapson, V.F., Boni Schnetzler, M., Pilch, P.F., Center, D.M. and Berman, I.S. (1988) Structural and functional characterization of the human T lymphocyte receptor for insulin-like growth factor I in vitro. J. Clin. Invest. 82, 950-957. Thiele, D.L., Kurosaka, M. and Lipsky, P.E. (1983) Phenotype
of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, Lleucine methyl ester. J. lmmunol. 131, 2282-2290. Thorsson, A.V. and Hintz, R.L. (1977) Specific ~:SI-somatomedin receptors on circulating human mononuclear cells. Biochem. Biophys. Res. Commun. 74, 1566-1573. Vaessen, L.M., Kreeftenberg, J.G., Heyse, P., et al. (1989) RIV-9: a mouse IgG3 anti-human CD3 monoclonal antibody with strong antigen modulating and T cell eliminating properties. Transplant. Proc. 21, 1026-1027. Van Buul-Offers, S. (1983) Hormonal and other inherited growth disturbances in mice with special reference to the Snell dwarf mouse. A review. Acta Endocrinol. Suppl. Copenh. 258, 1-47. Van Zoelen, E.J., van Oostwaard, T.M., van der Saag, P.T. and de Laat, S.W. (1985) Phenotypic transformation of normal rat kidney cells in a growth-factor-defined medium: induction by a neuroblastoma-derived transforming growth factor independently of the EGF receptor. J. Cell. Physiol. 123, 151-160. Weigent, D.A. and Blalock, J.E. (1991) The production of growth hormone by subpopulations of rat mononuclear leukocytes. Cell. Immunol. 135, 55-65. Weigent, D.A., Blalock, J.E. and LeBoeuf, R.D. (1991) An antisense oligodeoxynucleotide to growth hormone messenger ribonucleic acid inhibits lymphocyte proliferation. Endocrinology 128, 2053-2057. Zapf, J , Schmid, C. and Froesch, E.R. (1984) Biological and immunological properties of insulin-like growth factors (IGF) I and II. Clin. Endocrinol. Metab. 13, 3-30.
95
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02175
Effects of insulin-like growth factors and growth hormone on the in vitro proliferation of T lymphocytes R o n Kooijman
a, Mia Willems a, G e r T. Rijkers a, A d Brinkman c, Sylvia C. van Buul-Offers b, Cobi J. Heijnen a and Ben J.M. Zegers a
Department of Immunology, b Department of Endocrinology, UniversityHospitalfor Children and Youth "Het Wilhelmina Kinderziekenhuis", Utrecht, and c Department of Pathology I, Erasmus University, Rotterdam, The Netherlands (Received 4 November 1991) (Revised, received 23 December 1991) (Accepted 23 December 1991)
Key words: Insulin-like growth factor; Growth hormone; IGF-binding protein; Proliferation; T Lymphocyte; Mitogen
Summary The insulin-like growth factors I and II (IGF-I and IGF-II) promote proliferation and differentiation of many cell types. We report that recombinant IGF-I and IGF-II augment both the lectin- and anti-CD3-induced proliferation of human peripheral blood mononuclear ceils (PBMC) at concentrations proportional to their binding affinities. IGF-I and IGF-II also augmented the lectin-induced proliferation of purified T lymphocytes. Effects of IGF-I were found in cultures of T cells vigorously depleted for monocytes and supplemented with saturating concentrations of interleukin-1. The latter results indicate that the effect of IGF-I on the proliferation of T lymphocytes can occur independent of monocytes or monocyte-derived factors.
Introduction Growth hormone (GH) and the insulin-like growth factors (IGF-I and IGF-II) play a pivotal role in growth and development. The insulin-like growth factors comprise a family of peptides which promote proliferation and differentiation of various cell types (Froesch et al., 1985; Baxter, 1986). Although the distinction is certainly not absolute, it is assumed that IGF-II plays an im-
Correspondence to: R. Kooijman, Department of Immunology, Wilhelmina Kinderziekenhuis, P.O. Box 18009, 3501 CA Utrecht, The Netherlands.
portant role as a foetal growth factor, whereas IGF-I is more important for postnatal growth and development. Specific receptors for both growth factors have been described. The type I IGF receptor binds IGF-I and IGF-II with a high affinity, whereas the type II receptor binds IGF-II with a high affinity and IGF-I with a low affinity (Rechler and Nissley, 1985). It is generally accepted that the growth promoting effects of growth hormone, which become apparent after birth, are mediated by IGF-I (Isaksson et al., 1987). IGF-I is predominantly synthesized in the liver, but it can also be produced by a variety of tissues where it can act as an autocrine or paracrine growth factor (D'Ercole et al., 1984).
96 It is becoming evident that IGF-binding proteins may modulate many of the effects of IGFs in different tissues. Whereas most IGFs in the circulation are bound to a 150-kDa IGF-binding protein (IGFBP-3), the low molecular mass binding proteins are the main binding proteins in other body fluids. There is ample evidence that IGFBPs can modulate the growth promoting effects of IGFs. In various biological systems, IGFBPs have been shown to inhibit or potentiate IGF action (reviews by Hintz, 1990, Baxter, 1991). A role for IGF-I in the immune system is suggested by the expression of IGF-I in human macrophages and lymphoblastoid cell lines (Rom et al., 1988; Merimee et al., 1989), its ability to reverse thymic involution in diabetic rats (Binz et al., 1990), and its potent ability to activate neutrophils for superoxide anion production (Fu et al., 1991). Furthermore, both IGF-I and IGF-II might be involved in lymphocyte proliferation as suggested by the effects of both growth factors on the in vitro proliferation of lectin-activated lymphocytes (Schimpff et al., 1983; Tapson et al., 1988) and by the increased expression of type I and type II IGF receptors on phytohaemagglutinin (PHA) activated T cells (Thorsson and Hintz, 1977; Kozak et al., 1987; Tapson et al., 1988). Although IGF-I expression in a variety of cells and tissues can be regulated by factors other than GH, there are indications that GH can influence lymphocyte proliferation via secretion of IGF-I by lymphocytes. This is suggested by the observation that GH augments IGF-I secretion in Epstein Barr virus-transformed B cells (Merimee et al., 1989) and that the stimulatory effect of GH on the colony formation of Human T Cell Lymphoma Virus-I- and II-transformed T cells could be inhibited by antibodies against the type I receptor (Geffner et al., 1990). GH-induced IGF-I expression might play a pivotal role in the proposed interactions between GH and the immune system (Kelley, 1989). Results of in vitro studies on the effects of GH on the proliferation of peripheral blood mononuclear cells (PBMC) are contradictory, since both positive (Astaldi et al., 1973; Mercola et al., 1981) and negative (Kiess et al., 1983; Schimpff and Repellin, 1989) effects of GH have been reported. This may be due to the use of GH from different origins a n d / o r to the
culture conditions employed. Since the effects of GH may be mediated by IGF-I, it is important that experiments are performed under IGF-free conditions. The purpose of the present work was to determine whether IGF-I and IGF-II are involved in the in vitro proliferation of PBMC and in particularly of T cells. Since no dose-response relationship for IGFs using media without endogenous IGFs has been reported, we tested the effects of recombinant IGF-I and IGF-II on lymphocyte proliferation in serum-free medium and in fetal calf serum (FCS) that has been treated with dithiothreitol (DTT) in order to inactivate IGFs. We found that IGF-I and IGF-II augment the proliferation of PHA- and anti-CD3 activated PBMC and purified T cells at concentrations that are in accordance with their binding affinities.
Materials and methods
Cells
Human peripheral blood mononuclear cells (PBMC) were purified from heparinized blood from healthy adult donors by centrifugation on Ficoll/Isopaque (Pharmacia, Uppsala, Sweden) density gradients (1.077 g/ml) at 1000 x g for 20 min at room temperature. T cells were isolated from PBMC by rosetting with sheep erythrocytes that were pretreated with 2-aminoethyl-isothiouronium bromide (AET; Sigma, St. Louis, MO; Gmelig Meyling et al., 1977). This T cell fraction contained at least 90% CD3 positive cells and 1-2% monocytes as assessed by immunofluorescence cytometry using anti-CD3 (leu 4) and anti-CD14 (leu M3) as primary antibodies and fluorescein-conjugated goat-anti-mouse Ig as a secondary antibody (antibodies were obtained from Becton Dickinson, San Jose, CA). Residual monocytes in T cell-enriched preparations were removed by treatment with leucine methyl ester (Thiele et al., 1983). Chemicals and materials
Recombinant human growth hormone (Genotropin) was obtained from Kabivitrum (Stockholm, Sweden). Recombinant human IGF-I and IGF-II were kindly provided by Dr. Jeatran, Lilly
97 Research Laboratories, Indianapolis, IN. Human IGF-binding protein 1 (IGFBP-1) was purified from amniotic fluid (Drop et al., 1982). L-Leucine methyl ester and bovine serum albumin (BSA) were from Sigma (St. Louis, MO). The BSA did not contain any detectable IGF-activity as determined by radioimmunoassay. Recombinant IL-1/3 was a generous gift from Dr. A. Shaw, Glaxo Institute of Molecular Biology, Geneva, Switzerland. Normal human serum (NHS) was a pool from five normal adults and fetal calf serum (FCS) and was obtained from Gibco (Grand Island, NY). The FCS batches we used were selected for a low level of [3H]thymidine ([3H]dTd) incorporation by PBMC in the absence of mitogens (< 100 cpm). Growth factor-inactivated serum (SH-FCS) was prepared by treatment of FCS with 100 mM dithiothreitol (DTT; Sigma). After overnight dialyses against a 50-fold excess of phosphate-buffered saline, the formed sulfhydryl groups were alkylated with 5 mg/ml iodoacetamide (Sigma). DTT and iodoacetamide were removed by dialysis (molecular weight cutoff 8-10 kDa) against a 75-fold excess of phosphatebuffered saline which was repeated five times. A more detailed description of the procedure and further information about the composition of the inactivated serum has been presented by Van Zoelen et al. (1985). The anti-CD3 mAb RIV9 was a gift from Dr. J. Kreeftenberg, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands (Vaessen et al., 1989).
Cell culture and proliferation assays For PHA-induced proliferation, PBMC were cultured in round-bottom 96-well polysterene microtiter plates (Nunc, Kamstrup, Denmark) at a concentration of 2.7 X l0 s cells/ml in 150 /~1 medium containing RPMI 1640 (Gibco), 100 U / m l penicillin, 100/zg/ml streptomycin, 4 mM glutamine and 10% FCS (heat-inactivated: 45 min, 56°C) or 2% NHS (heat-inactivated). When the ceils were cultured in 10% SH-FCS or 0.2% BSA, the medium was supplemented with 12.5 /zg/ml transferrin and 30 nM sodium selenite. PBMC were stimulated with 15 /~g/ml phytohemagglutinin (PHA; HA15 Wellcome, Dartford, UK) and the T cells were activated with 40 /~g/ml PHA.
After 48 h of culture at 37°C in 5% C02, 100% relative humidity, the cells were pulsed with 37 kBq of [3H]dTd (1.65 TBq/mol; Amersham international, Amersham, UK) and then cultured for a further 18 h. When PBMC were activated with anti-CD3 (RIV9, 1:3000), they were cultured in flat-bottom wells at a concentration of 106 cells/ml (200 ~l total volume) for 72 h prior to the addition of [3H]dTd and were harvested 18 h thereafter onto glass fiber filters. The incorporated [3H]dTd was measured using standard scintillation procedures. The significance of observed differences between means were determined using Student's t-test.
Results
Effects of IGF-I and IGF-II on proliferation of human PBMC and T cells The effects of IGF-I and IGF-II on PHAactivated PBMC were tested using different culture conditions. When lymphocytes were cultured in 10% FCS, addition of GH, IGF-I or IGF-II did not influence the proliferation at concentrations that are consistent with their binding affinities for their receptors. Only high concentrations of IGF-I (i.e. 10 -7 M) increased proliferation. Since FCS contains high concentrations of IGF-I and IGF-II (Daughaday and Rotwein, 1989), we also tested the effects of growth factors using serum-free medium or medium supplemented with inactivated FCS (SH-FCS). The IGFs can be inactivated by reduction of disulfide bonds. As a result of inactivation with DTI', the IGF concentration in the serum decreases to less than 10 -11 M (Van Zoelen et al., 1985). Furthermore, at least certain IGF-binding proteins (IGFBPs) are inactivated by reduction (Hardouin et al., 1987), because the appropiate folding of the molecules depends on disulphide bonds (Brinkman et al., 1991). Replacement of FCS by SH-FCS supplemented with transferrin and sodium selenite resulted in a reduction of basal PHA-induced thymidine incorporation from 9500 to 5500 cpm (Fig. 1A). However, proliferation in this serum appeared to be 3 to 4 times higher than in a serum-free medium with bovine serum albumin (BSA) used at an equiva:lent protein concentration (data not pre-
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Fig. 1. Effects of recombinant human IGF-I (©), IGF-II (e) and G H (zx) on the proliferation of PBMC. A. PHA-activated cells were cultured for 66 h in 10% FCS (dashed lines) or 10% IGF-inactivated FCS (SH-FCS) supplemented with 12.5 / z g / m l transferrin and 30 nM sodium selenite (solid lines). B. Cells from the same donor were activated with an anti-CD3 mAb (RIV9) and cultured for 90 h in the medium with SH-FCS. Standard errors were below 10% of the mean. Asterisks indicate significant difference from control conditions (~ P < 0.05; * P < 0.005). These data are representative of four replicate experiments.
sented). The difference in cell proliferation between cultures performed in BSA or SH-FCScontaining medium could not be reduced by addition of extra transferrin (data not shown). Because of the higher proliferative response of the lymphocytes in SH-FCS, we preferred the use of this inactivated serum for most experiments. The PHA- and anti-CD3-induced proliferation of PBMC in 10% SH-FCS is augmented by IGF-I and IGF-II at concentrations that are in the order of magnitude of the free concentrations in serum and the K d of their receptors (Fig. 1A and B). The magnitude of the effects of IGF-I and IGF-II varied between individual donors, but the effects were always positive. For 10 -8 M IGF-I and IGF-II we found a mean increase in the proliferative response of 41% and 32%, respectively (Table 1). To test whether IGF-I and IGF-II can augment the proliferation of PBMC independently of other serum factors, we performed the same experiments in a serum-free medium which contains 0.2% BSA instead of SH-FCS. As depicted in Fig. 2, it appeared that IGF-I and
TABLE 1 MEAN EFFECTS OF IGF-I, IGF-II, G H A N D IGFBP-1 ON T H E P H A - I N D U C E D P R O L I F E R A T I O N O F PBMC Stimulation index + SD 10 9 M IGF-I 10 s M IGF-I 10 -9 M IGF-II 10 -8 M IGF-II 10 - s M GH 10 -7 M GH IGFBP-1 10 -8 M GH 10 -7 M GH
1.22+0.14 1.41 +0.27 1.15+0.16 1.32_+0.27 0.93+0.09 0.84_+0.08 0.70_+0.07 1.00+_0.04 0.94-+0.08
(n (n (n (n (n (n (n (n (n
= = = = = = = = =
7) 9) 5) 6) 6) 6) 6) 4) 4)
a
b b c J d
a The effects of IGF-I and IGF-II were measured in RPMI medium containing 10% SH-FCS. b Effects of G H in RPMI medium containing 10% SH-FCS. c Effects of IGF-binding protein 1 were studied on cells cultured in RPMI medium supplemented with 2% normal human serum (NHS). d Effects of G H measured in RPMI medium containing 0.2% BSA. The incorporation of [3H]dTd ranged between 3000 and 10000 cpm for cells cultured in RPMI containing NHS and between 1000 and 5000 cpm for the cells that were cultured in a medium with SH-FCS or BSA.
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[IGF] Fig. 2. Effects of recombinant human IGF-I ( o ) and IGF-II (e) on the proliferation of PHA-activated PBMC that were cultured for 72 h in RPMI containing 0.2% BSA, transferrin and selenite. The mean v a l u e s + S E M from quadruplicate wells are presented. These data are representative of three replicates.
IGF-II also augment the proliferation of PBMC in serum-free medium and that the dose-response curves for IGF-I and IGF-II were very similar to 40OO
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[hormone] Fig. 3. Effects of recombinant human IGF-I (o), IGF-II (e) and GH ( a ) on the proliferation of PHA-activated T lymphocytes. T cells were purified with AET-pretreated sheep erythrocytes and cultured in 10% SH-FCS supplemented with transferrin and sodium selenite in the presence of 4 0 / z g / m l P H A for 66 h in 96-well microtiter plates. Standard errors of the mean were below 5%. * Significantly different from control conditions (* P < 0.005). These data are representative of three replicate experiments.
the curves obtained using SH-FCS (see Fig. 1). The EDs0 of 0.12 nM for the effects of IGF-I in the serum-free system corresponds with the K d for the type I receptor on T cells (0.12 nM) as described by Tapson et al. (1988). The 10-fold higher IGF-II concentrations required to evoke the same effect as IGF-I reflect the affinity of IGF-II for the type I and type II receptors on lymphocytes (Kozak et al., 1987). In all of our experiments, the [3H]dTd incorporation was lower than 100 cpm when the cells were not treated with mitogen (data not presented). The effects of IGF-I and IGF-II on PHA-induced proliferation of purified T cells were much larger than those on PBMC (Fig. 3). IGF-I (10 nM) increased the proliferation of purified T cells obtained from different donors by 108 + 64% (n = 7) and IGF-II augmented proliferation by 78 + 21% (n = 3). The rosetted T cells, which could be in a partially activated state as a result of the rosetting procedure, did not respond to IGFs unless they were activated by PHA (data not shown). Although these results suggest that IGFs stimulate T cells directly, a role for monocytes in mediating the effects of IGFs cannot be excluded, because IGF-I receptor bearing monocytes (Stuart et al., 1991; manuscript in preparation) may contaminate the T cell fractions. To test whether IGF-I augments T cell proliferation via stimulation of monocyte-derived interleukin 1 (IL-1), we tested the effects of IGF-I on the proliferation of T cells in the presence of IL-1. It appeared that IGF-I still stimulated the proliferation of purified T cells in the presence of saturating levels IL-1 (Fig. 4A). These results indicate that the positive effect of IGF-I on T cell proliferation is not a secondary event due to an increased secretion of IL-1. To exclude other possible monocyte-mediated effects of IGF-I, the remaining monocytes in the T cell fraction were killed by incubation with leucine methyl ester. No monocytes could be detected in leucine methyl ester-treated T cells with a non-specific esterase staining (detection level 0.1%). As depicted in Fig. 4B, the effects of IGF-I on T cell proliferation were still observed in leucine methyl estertreated T cell preparations. When monocytes from the same donor, obtained by adherence onto plastic tissue flasks, were added to the cul-
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Fig. 4. Effects of IL-1 and monocytes on the augmentation of T cell proliferation by recombinant human IGF-I. A. Effects of IGF-I on PHA-induced T cell proliferation in the presence and absence of IL-1. All differences between the controls without IGF-I and the cultures with IGF-I are statistically significant (P < 0.05). Significant effects of IL-1 are indicated (11, P < 0.05). B. Effects of IGF-I on T cell proliferation in the absence and in the presence of different concentrations of monocytes. Open bars, control; stippled bars, 10 10 M IGF-I; cross-hatched bars, 10 - 9 M IGF-I; closed bars, 10 -s M IGF-I. Monocytes in the T cell fraction were killed by treatment with leucine methyl ester. Cell cultures with different concentrations monocytes were obtained by addition of monocytes that were isolated from PBMC by adherence onto plastic culture flasks. Statistically significant effects of IGF-I are indicated (* P < 0.05). The mean values + SEM from quadruplicate wells are presented. The data are representative of four experiments.
ture, we f o u n d t h a t th e positive effect of exogenous I G F - I on T cell p r o l i f e r a t i o n was r e d u c e d (Fig. 4B).
2000
o~
1500
1000
0
0
0.4
40 IGF-I
0.4
40
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IGF-II
Fig. 5. Inhibition of the proliferation promoting effect of recombinant IGF-I and IGF-II by IGF-binding protein 1 (IGFBP-1). PBMC were cultured for 66 h with 15 ~g/ml PHA in RPMI with 10% SH-FCS supplemented with transferrin and sodium selenite. The effects of different concentrations IGF-I and IGF-II were monitored in the presence (closed bars) and in the absence (open bars) of 5.4 nM IGFBP-1. The mean values _+SEM from quadruplicate wells are presented.
Effects of IGF-binding protein 1 In o r d e r to test t h e ability o f I G F B P - 1 to m o d u l a t e t h e effects o f I G F s on l y m p h o c y t e proliferation, we t e s t e d the effects of p u r i f i e d I G F B P - 1 o n the effects of I G F - I and I G F - I I on PHA-induced proliferation of PBMC. Figure 5 shows that t h e effects o f b o t h 0.4 n M r e c o m b i n a n t I G F - I and I G F - I I are a b r o g a t e d by 5.4 n M I G F B P - 1 . N o inhibitory effect o f I G F B P - 1 was f o u n d w h e n an excess o f I G F - I or I G F - I I was a d d e d . T o test t h e effects of I G F B P - 1 on t h e growth p r o m o t i n g effects o f native h u m a n I G F - I as p r e s e n t in serum, we c u l t u r e d P B M C in m e d i u m s u p p l e m e n t e d with 2 % n o r m a l h u m a n s e r u m ( N H S ) . It a p p e a r e d that a d d i t i o n o f 5.4 n M I G F B P - 1 i n h i b i t e d t h e m i t o g e n - i n d u c e d proliferation (Fig. 6). C o n c o m i t a n t a d d i t i o n o f I G F B P - 1 with excess I G F - I or I G F - I I ( 1 0 - 1 0 0 n M ) a b r o g a t e s t h e inhibitory effect of I G F B P - 1 (Fig. 6). T h e s e results show that the effects o f native an d r e c o m b i n a n t h u m a n I G F - I an d I G F - I I on t h e p r o l i f e r a t i o n o f P B M C can be i n h i b i t e d by I G F B P - 1 . A d d i t i o n of I G F B P - 1 to P B M C f r o m six d o n o r s c u l t u r e d in m e d i u m s u p p l e m e n t e d with 2 % N H S i n h i b i t e d the p r o l i f e r a t i o n of P H A act i v at ed l y m p h o cy t es f r o m 20 to 5 0 % with a
10l 15
BSA. Also lower concentrations (10 - u to 10 -9 M) were without any effect (data not presented). Similar to the results for IGF-I and IGF-II, GH was not able to induce proliferation of resting cells (data not presented).
O T.X
E
oe'l
10
tO °m
O O
Discussion
5
.=_ '10 0
0
10
100
IGF-I
10
100
r)M
IGF-II
Fig. 6. Inhibition of lymphocyte proliferation by IGF-binding protein 1 (IGFBP-1). P B M C were cultured in R P M I medium with 2% normal h u m a n serum (NHS) and 15 ~ g / m l P H A for 72 h. The effect of the binding protein was tested in the absence or the presence of different concentrations recombinant IGF-I and IGF-II. The concentrations native IGF-I and IGF-II in 2% NHS are 0.4 nM and 1.6 nM respectively. Open bars, control; closed bars, with 5.4 nM IGFBP-1. The mean values + SEM from quadruplicate wells are presented.
mean of 30% for six donors (Table 1). This indicates that a substantial part of the growth-promoting effect of human serum on PBMC is caused by IGFs.
Effects of recombinant human growth hormone In order to be able to measure IGF-I-mediated effects of GH, and synergistic effects of GH with other serum factors, the effects of growth hormone were determined in either 10% SH-FCS or 10% FCS. With either serum, we did not find a positive effect of GH on thymidine incorporation when the cells were stimulated with either PHA or anti-CD3 (Fig. 1A, B). Indeed, the effects of GH on PHA-induced proliferation were slightly negative (Fig. 1 and Table 1). Even though the proliferation of purified T cells was strongly augmented by IGF-I, it was not influenced by GH (Fig. 3). To avoid the possible effects of bovine GH from FCS, which binds only weakly to human growth hormone receptors (Leung et al., 1987), we also tested the effects of human GH in serum free medium containing 0.2% BSA. As depicted in Table 1, high concentrations of GH had no effect on the proliferation of PBMC in 0.2%
We investigated the effects of IGF-I and IGFII on the proliferation of human lymphocytes in various culture media. IGF-I and IGF-II had no effect on the proliferation of PBMC cultured in 10% FCS (Fig. 1A) or 2% NHS (Fig. 6) at concentrations which are in accordance with their usual dissociation c o n s t a n t s ( 1 0 - 1 0 t o 10 - 9 M ) , while significant positive effects were observed when 0.2% BSA or 10% SH-FCS was used. When FCS was replaced by SH-FCS, the addition of IGF-I or IGF-II could not restore the proliferation to the level in FCS, indicating that besides IGFs, the DTT treatment has also inactivated other factors that promote lymphocyte proliferation. The higher level of thymidine incorporation in SH-FCS compared to the level in BSA may be due to the presence of DTl'-resistant growth factors or lipids (Van Zoelen et al., 1985) which are important for lymphocyte proliferation (Spieker-Polet and Polet, 1981; Cuthbert and Lipsky, 1986). The ED50 for IGF-I on proliferation of PBMC in 0.2% BSA and 10% SH-FCS is equivalent to the K d of 0.12 nM for the type I IGF receptor on resting and PHA-activated T lymphocytes (Tapson et al., 1988). The 10 times higher concentration of IGF-II that is necessary to induce the same effect as IGF-I is in accordance with the relatively lower binding affinity of this factor for the type I and type II receptor. Although there are indications that IGF-II effects can be mediated by the type II receptor (Tally et al., 1987), it is generally accepted that most growth-promoting effects of IGF-II take place via binding to the type I receptor (Nissley and Rechler, 1984; Zapf et al., 1984). The contribution of native IGFs in human serum to the proliferation of PBMC was assessed by addition of an excess of the binding protein to PBMC cultured in R P M I / 2 % NHS. The inhibitory effect was donor-dependent and varied from 22 to
102
51% with a mean of 30% (Table 1). Therefore, we conclude that the IGFs are a relatively important serum factor for the proliferation of PBMC. When IGF-I or IGF-II was added to purified T cells cultured in R P M I / 1 0 % SH-FCS, we found an even greater increase in proliferation as compared to the effects on PMBC. Since the effects of IGF-I were also observed in a monocyte-free T cell culture supplemented with a saturating concentration of IL-1, we conclude that IGF-I can enhance T cell proliferation in the absence of monocytes. The stimulation index of IGF-I appeared to be inversely related with the percentage monocytes in the culture. This effect may be the result of secretion of IGFs or IGF-binding proteins by monocytes, which is currently a subject of investigation. Both IGF-binding and IGF-I proteins can be produced by cells of the immune system (Rom et al., 1988; Merimee et al., 1989; Baxter et al., 1991; Neely et al., 1991). Although the effects of IGFs are in the range observed in many other cell systems, it is still possible that the effects of IGF-I on T cells is a relatively strong effect on only a fraction of the T cells. A functional role for IGFs in lymphocyte proliferation is suggested by their positive effects on proliferation and upregulation of their receptors (Kozak et al., 1987; Tapson et al., 1988). This idea is supported by the results of Hansson et al. (1988) who showed that cytoplasmic IGF-I is present in part of the lymphocytes in the thymus and the spleen of rats, and by the observation of Baxter et al. (1991) that rat leukocytes produce IGF-I. These results and our observation that PHA-activated human T cells contain immunoreactive IGF-I (unpublished results) indicate that IGF-I might have an autocrine or paracrine function in the immune system. G H has been implicated to interact with the immune system in experimental animals. GH-deficient mice such as the Snell-Bagg dwarf mice are immuno-deficient (Baroni, 1967; Duquesnoy, 1972; Van Buul-Offers, 1983) and hypophysectomized rats have an impaired cellular and humoral immune response which can be restored by treatment with G H (Kelley, 1989; Edwards et al., 1991). Also in vitro experiments with HTLVtransformed human cell lines demonstrate that G H augments T lymphocyte colony formation
(Geffner et al., 1990). In the latter study, it was demonstrated that the effects of G H were mediated by locally produced IGF-I. In addition, Baxter et al. (1991) showed that G H stimulates the production of IGF-I in rat leukocytes. Reports on the effects of human G H on the in vitro proliferation of peripheral lymphocytes have yielded conflicting results. To avoid effects of contaminating substances and to allow the growth-promoting effect of G H to be mediated by IGF-I, we studied the effects of recombinant human G H on lymphocyte proliferation in 10% SH-FCS and 0.2% BSA. Although it has been reported that human G H can induce blastogenesis (Mercola et al., 1981) and thymidine incorporation in unstimulated PBMC (Kiess et al., 1983), we did not find an effect of recombinant human G H on the thymidine incorporation of resting lymphocytes, not even when employing the culture conditions as described by Kiess et al. (1983; data not shown). The effects of G H on PHA- and anti-CD3-induced proliferation, if any, were negative. Negative effects were also found by Schimpff et al. (1989) and Kiess et al. (1983). Although we used serum-free culture conditions, our results do not exclude a possible role for G H in lymphocyte proliferation, because G H has been shown to be produced by isolated PBMC (Weigent and Blalock, 1991). Indeed, the inhibition of G H production in rat leukocytes inhibits the proliferation (Weigent et al., 1991). Overall, our results indicate that IGFs are important growth factors for proliferation of human T cells and that the effects of IGF-I can occur in the absence of monocytes. More insight in the role of IGFs in the immune system can be obtained by further investigations on the effects of IGFs and by studying the expression of IGFs and their receptors in cells of the immune system.
Acknowledgements We are indebted to Drs. K.W. Kelley and S. Arkins (University of Illinois, IL) for their critical assessment of this paper, to Dr. A. Shaw (Glaxo Institute for Molecular Biology, Geneva, Switzerland) for kindly providing riLl/3 and to Dr. Jeatran (Lilly Research Laboratories, Indianapo-
103
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