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(Journal of Leukocyte Biology. 2001;70:297-305.)
© 2001 by Society for Leukocyte Biology

Early effects of insulin-like growth factor-1 in activated human T lymphocytes

Mariana G. Brocardo*, Roxana Schillaci{dagger}, Adriana Galeano{ddagger}, Martín Radrizzani*, Verónica White{dagger}, Anatilde González Guerrico*, Tomás A. Santa-Coloma* and Alicia Roldán{dagger}

* Instituto de Investigaciones Bioquímicas Fundación Campomar (IIB-UBA and IIBBA CONICET),
{dagger} Instituto de Biología y Medicina Experimental CONICET, and
{ddagger} Laboratorio de Patología Sanatorio Mater Dei, Buenos Aires, Argentina

Correspondence: Alicia Roldán, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina. E-mail: aroldan{at}dna.uba.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study evaluates the effects of insulin-like growth factor (IGF)-1 receptor (IGF-1R) down-regulation in stimulated T lymphocytes by investigating the expression of early activation proteins CD69, CD25, and interleukin (IL)-2. We found that IGF-1 does not modify CD69 expression but increases transcription and protein synthesis of CD25 and IL-2. The lowest level of IGF-1R detected after 15 min of activation suggested that the effects of IGF-1 occur at the initiation of cell activation. The activation of IGF-1R was confirmed by IGF-1R phosphorylation and increased phosphorylation of microtubule-associated protein kinase. We also detected the alternative IGF-1 transcripts Ea, with paracrine/autocrine regulation, and Eb, with endocrine regulation, in Jurkat cells and in quiescent T lymphocytes, and we detected IGF-1 protein in the culture medium after stimulation. These data suggest that the proliferative effects of IGF-1 on T lymphocytes include both autocrine/paracrine and endocrine processes.

Key Words: CD25 • IL-2 • IGF-1 receptor • IGF-1 mRNA • MAPK


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin-like growth factor (IGF)-1 is synthesized in numerous tissues and acts not only as a growth factor but also as a differentiation factor [1 ]. The way in which a tissue responds to IGF-1 depends on its differentiation state, its cell surface receptors, the cellular microenvironment, and how the IGF-1 molecule is delivered to its site of action. The production of IGF-1 also varies in different cell types. The transcription product of its gene consists of six exons. The N-terminal portion of exon 4 codes the E peptide, which is present only in the unprocessed hormone. In humans, the E peptide assumes one of two forms, depending on whether exon 4 is spliced to exon 5 (the Eb region) or exon 6 (the Ea region). IGF-1 protein translated from Ea mRNA has an autocrine/paracrine action in nonhepatic tissues, whereas IGF-1 derived from Eb mRNA is expressed mainly in the liver after stimulation by growth hormone (GH) [2 ].

In previous studies, our laboratory and others have demonstrated that IGF-1 stimulates the proliferation of human T lymphocytes [3 , 4 ]. This effect might be mediated during the early phases of activation, because we observed a down-regulation of IGF-1 receptors (IGF-1R) 1 h after the addition of mitogen [5 ]. As is found with insulin [6 ], epidermal growth factor [7 ], and IGF-1 [8 ], receptor-mediated endocytosis of a polypeptide hormone commonly occurs after the binding of the ligand to its specific receptor. Although this process is linked to the degradation of the hormone, it also plays a role in the mechanism by which these agents elicit their cellular responses [9 ].

Activation of T-lymphocyte receptors requires the cooperation of multiple signal transduction molecules, including the microtubule-associated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways [10 ], both of which are also activated by IGF-1R [8 ], making cross-talk possible between these receptors. On the other hand, the transcripts of several genes that are induced by T-cell activation encode membrane receptors and intracellular-signaling proteins that are expressed throughout the 2–3 weeks in which the process is accomplished [11 ]. The first early induced protein that is known to be expressed is CD69, the mRNA of which is detectable within 30 min. The CD69 protein itself is detectable in the cell membrane within 1 h [12 ]. Other early activation molecules are interleukin (IL)-2 and the {alpha}-subunit of its receptor (CD25), both essential for cell cycle progression and mitosis [13 ]. These two molecules are synthesized within hours of activation and before cell division.

Here we report the effects of IGF-1 addition and IGF-1R endocytosis on early expression of the membrane proteins CD69 and CD25, as well as the timing of IL-2 production in human peripheral-blood T lymphocytes and in the Jurkat T-cell line, during the first 12 h after activation. The activation of IGF-1R was confirmed by its receptor internalization together with activation of the MAPK pathway, which is dependent on IGF-1R internalization [8 ].

Finally, the rapid decrease in expression of IGF-1R after 15 min of activation in IGF-1-free medium suggests that T cells and T-cell lines might themselves produce IGF-1. Therefore, we examined the expression of the alternative transcripts Ea and Eb of IGF-1 in these cells, which should reflect the autocrine/paracrine effects of IGF-1 and its endocrine regulation, respectively. There is, however, the possibility that within the normal course of T-cell activation, IGF-1 receptors may be down-regulated independently of IGF-1 binding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Dulbecco’s modified Eagle’s medium containing 1 g/L of glucose, Ham’s F-12 medium, RPMI 1640 medium, fetal calf serum (FCS), and gentamicin was purchased from Gibco BRL Life Technologies Inc. (Gaithersburg, MD). Glutamine, 2-mercaptoethanol (2-ME), human transferrin (with no IGF-1 contamination, as measured in our laboratory), phorbol 12-myristate 13-acetate (PMA), and phytohemagglutinin (PHA)-P were purchased from Sigma Chemical Co. (St Louis, MO). Recombinant IGF-1 was kindly provided by J. P. Merryweather, Chiron Corp., Emeryville, CA, and recombinant human IL-2 teceleukin (TECINTM) was provided by the Biological Response Modifiers Program (National Cancer Institute, Bethesda, MD). Antibodies were purchased as follows: monoclonal antibody to IGF-1R ({alpha}-IR3) from Oncogene Science Inc. (Cambridge, MA); fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse, anti-phospho-extracellular kinase (ERK)-1/ERK-2, and anti-actin antibodies from Sigma; FITC-conjugated anti-human CD69 and phycoerythrin (PE)-conjugated CD25 from Caltag Laboratory (Burlingame, CA); PE- or peridinin chlorophyll protein (PerCP®)-conjugated anti-human CD3 from Becton Dickinson (San Jose, CA); monoclonal antiphosphotyrosine, 4G10, rabbit anti-insulin receptor substrate (IRS)-1 and anti-PI-3K antibodies from Upstate Biotechnology Industries (Lake Placid, NY); rabbit anti-IGF-1R {alpha}-subunit from Santa Cruz Biotechnology (Santa Cruz, CA); and horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) and alkaline phosphatase-conjugated anti-rabbit IgG from Promega (Madison, WI).

The quantitative determination of IL-2 concentrations was made using an enzyme-linked immunosorbent assay kit from Genzyme Diagnostic (Cambridge, MA).

Cells
The Jurkat T-leukemia cell line was maintained in RPMI 1640 medium supplemented with 5% FCS, as previously described [5 ]. When serum-free medium was used, the culture medium was changed for 16 h before stimulation to Dulbecco’s modified Eagle’s medium/Ham’s F-12 [1:1 (v/v)] (D:H), supplemented as described previously [5 ]. Thereafter, cells were stimulated with 10 ng/mL of PMA plus 1 µg/mL of PHA. For assays requiring 5 nM of IGF-1 or 60 IU/mL of IL-2, these proteins were added simultaneously with the stimuli.

Peripheral-blood mononuclear cells (PBMC) were obtained from the blood of healthy donors after their informed consent. T lymphocytes were isolated by rosetting with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, using a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). T-lymphocyte purity was 87 ± 2.34%, as demonstrated by flow cytometry. To achieve 96.5 ± 0.67% purity for reverse transcription (RT)-PCR detection of IGF-1 mRNA Ea and Eb assays, a human T-cell enrichment column (R&D Systems, Minneapolis, MN) was used, after treatment with sheep red blood cells. Cells were cultured in D:H medium and stimulated with 2 µg/mL of PHA. When appropriate, 5 nM IGF-1 or 60 IU/mL of IL-2 were added to the culture medium.

Flow-cytometric analysis
Expression of IGF-1R was evaluated by indirect immunofluorescence. Reactivity was determined in stimulated and unstimulated cells (106/mL), as described previously [5 ]. Briefly, cells were incubated for 30 min on ice with {alpha}-IR3 antibody (5 µg/mL), followed by anti-mouse FITC-conjugated antibody and finally with PE-conjugated anti-CD3. CD25 and CD69 expression was determined by direct immunofluorescence assay of the CD3-positive population.

Cells were examined for fluorescence intensity using FACScan cytometer (Becton Dickinson). A total of 104 cells/sample were analyzed. Background staining was evaluated in cells incubated with the FITC- or PE-conjugated isotype controls. Data analysis was performed using Lysys II software (Becton Dickinson).

RT-PCR analysis
Jurkat cells (5 x 106) or T lymphocytes (1 x 107) were harvested, and total RNA was prepared using the RNeasy kit (QIAGEN Inc., Valencia, CA). The RNA concentration was then determined for each sample, and 1 µg of total RNA was used in the RT reaction at 37°C for 60 min in a 25-µL reaction volume containing 50 mM Tris-HCl buffer (pH 8.3) supplemented with 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1.6 mM each deoxynucleotide triphosphate, 100 pmol of oligo (dT18) primer, and 200 U of Moloney murine leukemia virus reverse transcriptase (Promega Corp.). Transcribed cDNA fragments were amplified in a 20-µL reaction volume in 10 mM Tris-HCl (pH 8.3) supplemented with 50 mM KCl, 1.5 mM MgC2, 100 µM each deoxynucleotide triphosphate, 0.4 µM each primer, and 1 U of Taq DNA polymerase (Promega Corp.). Amplification was performed in a Perkin-Elmer 9600 thermal cycler (Perkin-Elmer Corp., Norwalk, CT). The primer sequences are shown in Table 1 .


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  		Table 1. Primers Used in RT-PCR

 
Amplification conditions for IL-2, CD25, and glycerol-3-phosphate dehydrogenase (G3PDH) mRNAs have been described by Zhou et al. [14 ]. Briefly, the reaction was subjected to two cycles of denaturation at 94°C for 2 min, annealing at 62°C for 2 min, and extension at 72°C for 5 min, followed by 28 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min. Amplified products were separated by electrophoresis on a 1.8% agarose gel and visualized by ethidium bromide staining and ultraviolet (UV) transillumination. The resulting mRNAs were evaluated by densitometric scanning using an HP4C scanner (Hewlett-Packard Co., Palo Alto, CA) and the NIH-IMAGE program (Sciogen Co., Detroit, MI). The values were expressed as ratios of arbitrary densitometric units of IL-2 or CD25 versus units of the G3PDH control band.

Amplifications of IGF-1 mRNAs Ea and Eb were performed separately under the conditions described by Swoli et al. [15 ], as follows: 40 cycles of denaturation at 94°C for 15 s, annealing at 62°C for 15 s, and extension at 72°C for 30 s. A denaturation step at 94°C for 5 min and an extension step at 72°C for 5 min were added to the initial and final cycles, respectively. Amplified products were separated by electrophoresis on a 1.8% agarose gel and visualized by ethidium bromide staining and UV transillumination.

A 100-bp ladder (Gibco BRL Life Technologies Inc.) was used to verify the sizes of the PCR products.

Western blot analysis
For determination of MAPK, T lymphocytes and Jurkat cells were stimulated for 5 min in D:H medium with 1 µg/mL of PHA alone and of PHA + 10 ng/mL of PMA, respectively, in the presence or absence of 5 nM IGF-1. Cells were lysed in ice-cold buffer [137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% Nonidet P-40, 10% glycerol, 2 mM EDTA, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM Na3VO4, 2 mM aminoethylbenzenesulfonyl fluoride, and 10 mg/mL of leupeptin in 20 mM Tris-HCl (pH 7.6)]. Cell lysates were centrifuged, the supernatants were collected, and protein concentrations were determined using the Bradford method, with bovine serum albumin as the standard. Next, 80 µg of protein were denatured in Laemmli buffer containing 200 mM 2-ME, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels, and electroblotted onto 0.45-µm-pore-size nitrocellulose membranes (Millipore, Bedford, MA). The blots were blocked with 2.5% nonfat milk and 0.1% Tween 20 in phosphate-buffered saline, incubated with anti-phospho-ERK-1/ERK-2 antibodies (1:5,000), washed, and incubated with horseradish peroxidase-conjugated anti-mouse IgG. RNA was detected using an enhanced chemiluminescence assay (Amersham, Arlington Heights, IL). For semiquantification of the proteins, the same blots were incubated with anti-actin antibody (1:100) and then visualized with alkaline phosphatase-conjugated anti-rabbit IgG, using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as chromogenic substrates (Gibco BRL). The values were expressed as the ratio of arbitrary densitometric units of phospho-ERK-1/ERK-2 versus units of actin control band.

Immunoprecipitation and immunoblotting
For immunoprecipitation, the cells were stimulated for 3 min and lysed as described above. Then 450-µg of protein was incubated in lysis buffer with 1.5 µg of the IGF-1R antibody {alpha}-IR3 at 4°C overnight. The antibody was adsorbed to protein-A/G agarose (Santa Cruz Biotechnology) for 2 h at 4 °C, and the immunocomplex was washed with buffer containing 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1% Nonidet P-40, 1 mM Na3VO4, and 1 mM aminoethylbenzenesulfonyl fluoride. Samples were then denatured in Laemmli buffer containing 200 mM 2-ME, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 7.5% gels, and electroblotted as described above. The blots were blocked and incubated with polyclonal IGF-1R {alpha}-subunit antibody (1:100) or anti-phosphotyrosine antibody (1:1,000), followed by horseradish peroxidase-conjugated anti-mouse IgG. Proteins were detected using enhanced chemiluminescence (Amersham). A similar procedure was used for the immunoprecipitation of IRS-1 (2 µg of antibody/sample), for PI-3K detection using anti-PI-3K (1:3,000), and for IRS-1 detection using anti-IRS-1 antibody (1:500).

Statistical analysis
Results are presented as means ± SE. Statistical analysis was based on Student’s t-test for paired comparisons of controls and treated samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of IGF-1 on the expression of immediate- and early-activation molecules induced by stimulation of T lymphocytes
To ascertain the temporal relationship between IGF-1R down-regulation and the early protein expression induced after T-lymphocyte stimulation, the kinetics of IGF-1R endocytosis during the first hour after stimulation was studied in human peripheral-blood T lymphocytes. Decreased IGF-1R was observed after 15 min of stimulation (Fig. 1 ). The addition of IGF-1 accelerated and increased this process, as observed in a previous study [5 ]. The same effect on IGF-1R expression was detected in activated T cells cultured in RPMI 1640 with 5% FCS (data not shown).



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  		Figure 1. Effects of activation and IGF-1 on the time-dependent expression of IGF-1R in stimulated peripheral-blood T lymphocytes. Cells were cultured in D:H medium and stimulated with PHA. Each solid line represents an independent donor. Dashed lines with matching symbols correspond to cells of the same donor in medium containing IGF-1.

 
Next the effect of IGF-1 on the surface expression of CD69 and CD25 in those cells was investigated during the period from 1 to 12 h after mitogen activation in medium lacking FCS. After 12 h of stimulation in the presence of IGF-1, CD25 and CD69 proteins were both expressed in the same cells, as shown in the dot blots in Figure 2 . However, addition of IGF-1 to the culture medium increased expression of CD25 but not of CD69 (Fig. 3 ). The lack of any IGF-1 effect on CD69 was observed in the number of CD69-positive cells, as well as in the number of receptors per cell (mean fluorescence) at any time between 0 and 12 h after activation (data not shown). Meanwhile, the effect on CD25 was observed at 12 h, when the expression of the protein was detected (Table 2 ).



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  		Figure 2. Representative dot blots of 12-h-stimulated peripheral-blood T lymphocytes. Cells were doubly stained for CD25 and CD69, as described in Materials and Methods, and numbers correspond to the percentages of positive cells in each quadrant: (a) isotype control; (b) stimulated cells; (c) stimulated cells in the presence of IGF-1.

 


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  		Figure 3. Representative histograms of 12-h-stimulated peripheral-blood T lymphocytes. Cells were doubly stained, as described in Materials and Methods. (a) CD69 expression; (b) CD25 expression. In both graphs, thin lines define the isotype control area, thick lines define the cultures with IGF-1, and gray areas define the cultures without IGF-1.

 

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  		Table 2. Expression of CD25 after T Lymphocyte Stimulation with or without IGF-1

 
The activation of T lymphocytes is known to first induce IL-2 gene transcription, followed by an increase in the mRNA encoding CD25, both being up-regulated by IL-2 [13 ]. Although we did not detect CD25 in the membranes of cells after 6 h of stimulation, its mRNA was already expressed at that time [16 ]. Thus, it can be seen in Figure 4 that, within 6 h of stimulation of the T cells, the transcription of CD25 was increased when IL-2 or IGF-1 was added to the culture medium, whereas neither cytokine affected quiescent cells (data not shown). These data confirm that IGF-1 up-regulates the expression of both CD25 mRNA and CD25 protein [17 ].



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  		Figure 4. Effects of IGF-1 and IL-2 on CD25 mRNA expression in PHA-stimulated T lymphocytes. RT-PCR analysis of CD25 (550 bp) and G3PDH (956 bp) mRNAs was performed. RT-PCR of 1 µL of total RNA from peripheral-blood T lymphocytes, nonactivated and activated in the absence or presence of IGF-1 or IL-2. Right lane, 100-bp DNA ladder. Quantification of each band was calculated as the average of triplicate readings from the same experiment. The values are expressed as the ratio of arbitrary densitometric units of CD25 versus units of G3PDH. *, P < 0.05 when compared with activated cells without cytokines. One experimental result is representative of three experiments.

 
IGF-1 regulation of IL-2 transcription and protein synthesis
Jurkat cells have long served as a useful human T-cell model to study the transcriptional regulation of genes encoding IL-2 and CD25. Stimulation of these cells with PHA plus PMA mimics the effect of T-cell-receptor activation [18 ]. Because IGF-1 had the same effect as IL-2 on CD25 mRNA expression, the effect of both cytokines on IL-2 mRNA levels was investigated in activated Jurkat cells and T lymphocytes. Figure 5a and b show that, after 6 h of stimulation of T lymphocytes and 4 h of stimulation of Jurkat cells, IGF-1 increased IL-2 mRNA expression to the same levels as did IL-2 itself. A similar effect was observed in T lymphocytes after 6 h of PHA treatment (Fig. 5b) , although in this case the effect of IL-2 was greater in all three experiments. The cytokines did not have any effect on nonstimulated Jurkat cells or T lymphocytes (data not shown).



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  		Figure 5. Effects of IGF-1 and IL-2 on IL-2 mRNA expression. RT-PCR analysis of IL-2 (458 bp) and G3PDH (956 bp) mRNA expression was performed in (a) PMA + PHA-stimulated Jurkat cells or (b) PHA-stimulated T lymphocytes cultured with or without IGF-1 or IL-2. Quantification of each band was calculated as the average of triplicate readings from the same experiment. The values are expressed as the ratio of arbitrary densitometric units of IL-2 versus units of G3PDH. *, P < 0.05, **, P < 0.01 when compared with activated cells without cytokines. One experimental result is representative of three experiments.

 
The effect of IGF-1 on IL-2 secretion was also investigated. The results confirmed a correlation between the increase in IL-2 mRNA and IL-2 secretion by both T lymphocytes and Jurkat cells (Table 3 ). Because Calvo et al. described a faster transcription of the IL-2 gene in Jurkat cells than in T lymphocytes [19 ], samples of Jurkat cells were taken after 4 and 8 h of activation, and samples of T cells were taken after 6 and 12 h of activation. The increase induced by IGF-1 in T cells was greater at 6 h than at 12 h (107.66 ± 10.36% vs.57.66 ± 2.72%, respectively). However, although IL-2mRNA expression by Jurkat cells was higher than that of T lymphocytes, the relative increase induced by IGF-1 was much lower (only 18%) after 8 h of activation, with none at 4 h. This may be a consequence of the PMA requirement for Jurkat cell activation [18 ]; PMA has such a powerful stimulatory effect that any further increase by IGF-1 stimulation would be difficult to achieve.


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  		Table 3. Effect of IGF-1 on IL-2 Production by Stimulated T Lymphocytes and Jurkat Cells

 
IGF-1 effect on activation of IGF-1R, MAPK, and PI-3K
To confirm the involvement of IGF-1R down-regulation in T-cell activation, we investigated the effect of IGF-1 addition on the divergent signaling pathways of the Shc proteins and IRS-1. We studied first the MAPK phosphorylation of ERK-1 and ERK-2 (the Shc pathway) and second the association of IRS-1 with PI-3K. As shown in Figure 6 , IGF-1 increased phosphorylation of ERK-1 and ERK-2 by 60% in stimulated lymphocytes, but it had no observable effect on Jurkat cells. This could be a consequence of the high levels of ERK-1 and ERK-2 phosphorylation after activation in the Jurkat cells [21-fold higher than in lymphocytes (Fig. 7 )], which would make difficult any further increase in response. The same phenomenon was observed for the production of IL-2 (Table 3) .



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  		Figure 6. Effects of IGF-1 on MAPK phosphorylation in (a) PMA + PHA-stimulated Jurkat cells and (b) PHA-stimulated T lymphocytes, cultured with or without IGF-1. Western blots were probed with anti-actin antibodies and with antibodies against phosphorylated MAPK. The bar graphs at the bottom were derived from the quantitation of the phosphorylated bands in three independent experiments. The values are expressed as the ratio of arbitrary densitometric units of phosphorylated ERK-1 and ERK-2 versus units of actin. *, P < 0.05 when compared with activated cells without cytokines.

 


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  		Figure 7. Detection by Western blotting of phosphorylated MAPK in PHA-stimulated T lymphocytes (TL) and PMA + PHA-stimulated Jurkat cells. The bar graph at the bottom was derived from the quantitation by arbitrary units of the phosphorylated bands. The values are expressed as ratios of arbitrary densitometric units of phosphorylated ERK-1 and ERK-2 versus units of actin.

 
The results were not as clear when the activation of the IRS-1 pathway was investigated (Fig. 8 ). This may reflect very low levels of IRS-1 or the absence of this pathway, which is independent of receptor internalization [8 ]. In stimulated and nonstimulated lymphocytes, in the presence or absence of IGF-1, neither IRS-1 nor PI-3K were detected by immunoblotting after inmunoprecipitation of IRS-1 from whole-cell detergent extracts (data not shown). When whole-lymphocyte detergent extracts were immunoprecipitated with antibody directed against the {alpha}-subunit of IGF-1R (Fig. 9 ), a phosphorylated band of 98 kDa, which corresponds to the molecular mass of the ß-subunit of IGF-1R [8 ], was observed only in cells stimulated in the presence of IGF-1.



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  		Figure 8. Detection by Western blotting of IRS-1 in T lymphocytes (TL), PBMC, and 3T3-L1 fibroblasts (3T3).

 


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  		Figure 9. Effect of IGF-1 on IGF-1R phosphorylation in nonstimulated and PHA-stimulated T lymphocytes, cultured with or without IGF-1. Whole-cell detergent extracts were immunoprecipitated with antibody directed against {alpha}-IGF-1R and then immunoblotted with {alpha}-IGF-1R antibody or phosphotyrosine antibody (P-Tyr). One experimental result is representative of three experiments.

 
IGF-1 synthesis in nonactivated cells
The rapid endocytosis of IGF-1R induced by the activation of cells cultured in medium without IGF-1 suggests that the cells themselves may synthesize and release IGF-1. Jendraschak et al. [20 ] observed variable levels of IGF-1 mRNA Ea and Eb in human PBMC and did not detect any IGF-1 transcripts in 2 of 14 samples. Neither isoform has been detected previously in Jurkat cells. Figure 10 shows that PBMC and Jurkat cells expressed both IGF-1 mRNA isoforms. T lymphocytes obtained from the same samples of PBMC with a T-cell enrichment column expressed IGF-1 mRNA Ea and Eb (Fig. 10) . However, not all samples showed both isoforms; in three of nine samples of PBMCs, only the Ea isoform was detected. Protein synthesis of IGF-1 by stimulated Jurkat cells was confirmed by radioimmunoassay in an FCS-free culture medium. After stimulation of the cells for 1 h, the concentration of IGF-1 in the medium was 589.3 ± 45.5 pg/mL.



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  		Figure 10. (Top and middle) Detection of IGF-1 mRNA Ea and Eb, respectively, in unstimulated PBMC and Jurkat cells using RT-PCR. Right lane, 100-bp DNA ladder. Each isoform was run separately at two different concentrations: Bands 1 and 4 were dilutions (1:2 v/v) of the samples seeded in bands 2 and 3. One experimental result is representative of nine experiments. In three of nine PBMC samples, only the Ea isoform was detected (data not shown). (Bottom) Detection of Eb and Ea in nonstimulated T lymphocytes obtained from the same PBMC with a T-cell enrichment column as described in Material and Methods from the sample shown in the panels above. positive control, an Ea cDNA of 700 bp inserted into the pKT218 plasmid (ATCC PHIGF-1).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The down-regulation of IGF-1R that we observed after 15 min of activation and the increase in MAPK phosphorylation after 5 min coincided with the time course of IGF-1 receptor internalization and MAPK activation in Chinese hamster ovary cells transfected with human IGF-1R, as described by Chow et al. [8 ], and confirms the involvement of IGF-1 in T-cell activation. Our results also show that, although IGF-1 does not control the synthesis of CD69 (the first protein expressed in stimulated T cells after 1 h of activation [12 ]), it does enhance the transcription of IL-2, which is induced after 2 h of activation, before CD25 [21 ]. Therefore, it is probable that IGF-1 influences all the genes expressed in the second stage of activation and not in the first one, in accordance with the classification of Ulman et al. [11 ].

On the other hand, the subsequent increased expression of IL-2 and the expression of its receptor, CD25, are in agreement with the observation of Kooijman et al. [22 ] that IGF-1 induces an increment in IL-2 production after 12 h of PBMC stimulation, which also provides an explanation for the shorter time spent by the cells in the G1 phase of the cell cycle in the presence of IGF-1, which we reported previously [23 ]. The high levels of ERK-1 and ERK-2 observed after Jurkat stimulation (Fig. 7) explain not only the higher production of IL-2 by those cells but also the low effect of IGF-1, and they confirm previous observations that with maximum stimulus it is very difficult to observe any effect of IGF-1 on T-cell stimulation [17 ].

Finally, the rapid internalization of IGF-1R seen after the stimulation of cells cultured in serum-free medium and also reported in Jurkat cells in earlier research [5 ], together with the presence of IGF-1 mRNA and detection of the hormone in the culture medium after 1 h of stimulation, suggest that an autocrine-/paracrine-stimulated release of IGF-1 is induced immediately after cell activation. However, it is possible that, in the normal course of T-cell activation, IGF-1 receptors are down-regulated independently of IGF-1 binding. Baxter et al. [24 ] have already suggested that accessory cells can release IGF-1, and both or either IGF-1 mRNA has been observed in human PBMC [20 ] and in murine macrophages [25 ].

In conclusion, our results suggest that the proliferative effects of IGF-1 in activated T lymphocytes are exerted mainly through the endocytic/Shc-MAPK pathway [8 ]. T lymphocytes may be regulated in an autocrine/paracrine manner by the release of this protein by T lymphocytes and accessory cells, but this requires further confirmation. It is also possible that there is endocrine regulation by GH, through the expression of IGF-1 mRNA Eb in T lymphocytes and perhaps by IGF-1 release in the liver. The presence of both Ea and Eb alternative transcripts could explain, at least partially, the lack of immune dysfunction in GH-deficient subjects [26 ], probably resulting from compensation of the Eb transcript with the Ea transcript, which causes paracrine/autocrine secretion of IGF-1 as was recently observed by Yakar et al. [27 ]. Furthermore, new therapies using IGF-1 and GH in HIV-infected patients must be considered [28 ], as well as the possible involvement of IGF-1 in diseases in which its level in serum is decreased, as occurs in malnutrition [29 , 30 ] and in immunodeficiency [31 ].


   ACKNOWLEDGEMENTS
 
This work was supported by a grants from Agencia Nacional de Promoción Científica y Tecnológica (BID 802/OC-AR, PMT-PICT 0486), the CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina, PEI 61/97) and Fundación Alberto Roemmers.

We are grateful to Dr. Damasia Becú-Villalobos from the Laboratory of Hypophysis Regulation of IBYME for the IGF-1 radioimmune assay, to Dr. Elsie M. Eugui and Anthony C. Allison for thoughtful review of this manuscript, from whom we have received continuous assistance, and to Dr. Pedro Di Spagna for supplying the blood samples from donors at the Hospital Militar Central Cosme Argerich.


   FOOTNOTES
 
M. G. Brocardo and R. Schillaci contributed equally to this work.

Received January 14, 2000; revised April 16, 2001; accepted April 17, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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