* Instituto de Investigaciones Bioquímicas Fundación Campomar (IIB-UBA and IIBBA CONICET),
Instituto de Biología y Medicina Experimental CONICET, and
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
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: CD25 • IL-2 • IGF-1 receptor • IGF-1 mRNA • MAPK
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
-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.
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-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 -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 -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
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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 -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 -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.
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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.
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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
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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.
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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.
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Table 3. Effect of IGF-1 on IL-2 Production by Stimulated T Lymphocytes and
Jurkat Cells
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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.
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-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 -IGF-1R and then immunoblotted with -IGF-1R
antibody or phosphotyrosine antibody (P-Tyr). One experimental result
is representative of three experiments.
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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).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 ].
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.
Received January 14, 2000; revised April 16, 2001; accepted April 17, 2001.
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chain expression in human lymphocytes Immunol. Cell Biol. 73,340-345
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