Interleukin-2 receptor-gamma -dependent endocytosis depends on biotin in Jurkat cells

Rocio Rodriguez-Melendez1, Gabriela Camporeale1, Jacob B. Griffin1, and Janos Zempleni1,2

Departments of 1 Nutritional Science and Dietetics and 2 Biochemistry, University of Nebraska at Lincoln, Lincoln, Nebraska 68583


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biotin has been credited with having beneficial effects on immune function despite observations that biotin supplementation causes decreased secretion of interleukin-2. Here this paradox was addressed by determining whether receptor-dependent internalization of interleukin-2 by immune cells depends on biotin. Theoretically, this would be consistent with both decreased net secretion of interleukin-2 by biotin-supplemented cells (causing increased endocytosis) and beneficial effects of biotin on immune function (causing increased receptor signaling). Jurkat cells were cultured in biotin-defined media (25, 250, or 10,000 pM). Secretion of interleukin-2 correlated negatively with biotin supply, but transcriptional activity of the interleukin-2 gene correlated positively with biotin supply, suggesting that decreased secretion of interleukin-2 by biotin-supplemented cells was not caused by decreased gene expression. Expression of the interleukin-2 receptor-gamma gene was greater at 10,000 pM than 25 pM biotin, mediating increased endocytosis of interleukin-2 in biotin-supplemented medium. Inhibition of endocytosis by genistein and overexpression of interleukin-2 receptor-gamma abolished the effect of biotin. These findings suggest that endocytosis of interleukin-2 depends on biotin.

cytokines; gene expression; propionyl-CoA carboxylase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TH1 lymphocytes respond to stimulation by antigens with transcription of the gene encoding interleukin (IL)-2 (17). After secretion into the extracellular space, IL-2 binds to IL-2 receptors (IL-2R) located on the surface of T and B cells, natural killer cells, and some myeloid cells (17). Three distinct IL-2 receptors have been identified in human cells: IL-2Ralpha , IL-2Rbeta , and IL-2Rgamma (17, 37). IL-2Rbeta and IL-2Rgamma are expressed constitutively; in contrast, IL-2Ralpha is expressed only in response to stimulation of immune cells (39). IL-2R may associate on the cell surface to form the following heteromers: 1) high-affinity receptor: one molecule each of IL-2Ralpha , IL-2Rbeta , and IL-2Rgamma (Kd for IL-2 = 0.01 nM); 2) intermediate-affinity receptor: IL-2Rbeta and IL-2Rgamma (Kd = 1 nM); and 3) low-affinity receptor: IL-2Ralpha (Kd = 10 nM) (37, 39).

Binding of IL-2 to IL-2R triggers intracellular signaling cascades, which involve phosphorylations of tyrosine kinases such as Jak3 and signal transducers such as Stat5 (17, 37). These cascades lead to growth and differentiation of immune cells (17, 31, 37). Signal transduction depends on IL-2Rgamma , suggesting a pivotal role of IL-2Rgamma in the human immune response (37, 39).

Ultimately, IL-2-IL-2R complexes are endocytosed and degraded (14, 15, 25, 36, 38, 41, 42) to avoid excessive stimulation of the immune system by IL-2. IL-2Ralpha is endocytosed less extensively than IL-2Rbeta and IL-2Rgamma , and the majority of endocytosed IL-2Ralpha is recycled to the cell surface (15, 25, 36, 41, 42).

Numerous studies have provided evidence for an essential role of the vitamin biotin in immune function (3, 7, 18, 28-30). In previous investigations we determined whether beneficial effects of biotin supplementation on immune function are mediated by increased secretion of IL-2. Surprisingly, we observed (21, 43) that secretion of IL-2 was negatively correlated with cellular biotin supply in freshly isolated human lymphocytes and in Jurkat cells. In the present study we hypothesized that expression of IL-2R (and thus endocytosis of IL-2) is dependent on biotin concentrations in immune cells. This hypothesis is consistent with decreased (apparent) secretion of IL-2 by biotin-supplemented cells (because of increased endocytosis) despite the beneficial effects of biotin supplementation on immune function (because of increased IL-2 signaling activity). A human lymphoid cell line (Jurkat cells) that expresses IL-2Ralpha and IL-2Rgamma (12, 14) was used to test this hypothesis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and culture conditions. Jurkat cells (clone E6-1) were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured (5% CO2 at 37°C in humidified atmosphere) in the following biotin-defined media for at least 5 wk before sample collection: 25 pM biotin (denoted "deficient"), 250 pM biotin ("physiological"), or 10,000 pM biotin ("pharmacological"); culture medium was replaced with fresh medium every 48 h. Media were prepared as described previously (21); biotin concentrations in media were confirmed by an avidin-binding assay (22) with modifications (21).

Biotin concentrations in media were chosen on the basis of the following lines of reasoning. Two hundred fifty picomolar biotin equals the physiological concentration of biotin in plasma from healthy adults (23). Twenty-five picomolar biotin is more than two standard deviations below the mean physiological concentration in normal plasma (23) and thus equals a deficient concentration of biotin. Ingestion of a typical biotin supplement providing 25 times the adequate intake of biotin for adults (26) is associated with plasma concentrations of ~10,000 pM biotin in healthy adults (43); thus this concentration represents a pharmacological concentration of biotin in plasma.

Cell viability was monitored at timed intervals by using Trypan blue as previously described (44); viability of cells was 98-100%. Consistent with this finding, previous studies showed that activities of the enzyme caspase-3 (marker for apoptosis) are below the limit of detection when Jurkat cells are incubated in media containing 25-10,000 pM biotin (21).

Propionyl-CoA carboxylase activity. Biotin serves as a coenzyme for propionyl-CoA carboxylase (PCC). Activities of PCC were quantified in cell extracts to confirm that intracellular biotin concentrations correlated with the biotin concentrations in media. The carboxylase assay quantifies the binding of radioactive bicarbonate to propionyl-CoA, catalyzed by PCC in samples of lysed cells. The assay was conducted as described previously (46) with modifications (21).

Secretion of IL-2. Secretion of IL-2 was induced by incubating 106 Jurkat cells with 50 µg/l of phorbol 12-myristate 13-acetate (PMA) and 2 mg/l of phytohemagglutinin (PHA) in a final volume of 260 µl as described previously (21). Times of stimulation varied among experiments and are specified in RESULTS. Cell-free medium supernatant was collected by centrifugation and analyzed for IL-2 with a commercial ELISA (hIL-2 EAISA; Biosource, Camarillo, CA) as described previously (21).

In the present study we hypothesized that the increased (apparent) secretion of IL-2 by biotin-deficient cells (21, 43) is caused by decreased endocytosis of IL-2 by these cells. Thus inhibitors of endocytosis should theoretically abolish effects of biotin on net secretion of IL-2. To test this hypothesis, cells were stimulated with PMA and PHA for 6 h as described above in the presence of 250 µM genistein. Inhibition of tyrosine kinases by genistein is known to inhibit endocytosis of IL-2 (42). Cell-free supernatants were collected and assayed for IL-2 as described above.

Reverse transcriptase polymerase chain reaction. mRNA encoding IL-2, IL-2Ralpha , IL-2Rgamma , and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; control) were quantified by reverse transcriptase polymerase chain reaction (PCR) in analogy to our previous studies (45). The following customized primers were used for PCR (Integrated DNA Technologies, Coralville, IA): 1) 5'-ATG TAC AGG ATG CAA CTC CTG TCT TGC-3' and 5'-AGT CAG TGT TGA GAT GAT GCT TTG ACA-3' for human IL-2 (GenBank accession number X01586); 2) 5'-ATG GAT TCA TAC CTG CTG ATG TGG GGA-3' and 5'-CTA GAT TGT TCT TCT ACT CTT CCT CTG-3' for human IL-2Ralpha (GenBank accession number XM043150); 3) 5'-TGA CGC CCA ATG GGA ATG AAG ACA CCA-3' and 5'-TCT TCA GGG TGG GAA TTC GGG GCA TCG-3' for human IL-2Rgamma (GenBank accession number AH002843); and 4) 5'-ACC ACA GTC CAT GCC ATC ACT GCC ACC-3' and 5'-TCC ACC ACC CTG TTG CTG TAG CCA AAT-3' for human G3PDH (2). Equal volumes (10 µl) of PCR product from each sample were separated on 1.5% agarose gel, stained with ethidium bromide, and analyzed with the Kodak EDAS 290 documentation and analysis system (Rochester, NY). Only values from within the exponential phase of PCR amplification (typically <48 PCR cycles) were considered for data analysis.

Reporter gene constructs. The following constructs were used to model effects of biotin on 5'-flanking regions of genes encoding IL-2 and IL-2Rgamma . 1) A construct of the regulatory region of the IL-2 gene (spanning 321 bases upstream of the transcription start site) linked to the luciferase gene [denoted p(-321)IL2-Luc] was provided by L. P. Freedman (Memorial Sloan-Kettering Cancer Center, New York, NY; Ref. 1). The regulatory elements of the IL-2 gene are located within ~300 bases upstream of the transcription start site (11, 16). 2) A construct of the regulatory region of the IL-2Rgamma gene (spanning 600 bases upstream of the transcription start site) linked to the luciferase gene (denoted pPBgamma 600) was provided by H. Asao (Tohoku University School of Medicine, Sendai, Japan; Ref. 27). The regulatory elements of the IL-2Rgamma gene are located within 600 bases upstream of the start site (27). 3) A construct of the RSV promoter linked to beta -galactosidase gene (denoted RSV beta gal) was used as control for transfection efficiency (provided by B. R. White, University of Nebraska-Lincoln, Lincoln, NE).

Fifteen million cells were cotransfected with luciferase constructs [p(-321)IL2-Luc or pPBgamma 600] and control construct (RSV beta gal) with SuperFect (Qiagen, Valencia, CA) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were stimulated with 50 µg/l of PMA and 2 mg/l of PHA for 6 h to induce expression of reporter genes. Luciferase activity was assayed by LucLite Plus (Packard, Boston, MA), according to the manufacturer's instructions, with a Top Count NXT (Packard). beta -Galactosidase activity was assayed with a commercial assay kit (Promega, Madison, WI) and an Emax Microwell Plate Reader (Molecular Devices, Sunnyvale, CA). All data were normalized for transfection efficiency, as judged by beta -galactosidase activity in response to transfection with RSV beta -gal.

Western blots. Effects of biotin supply on abundance of the receptor proteins IL-2Ralpha and IL-2Rgamma were investigated with Western blot analysis. Cellular proteins (from 7.5 × 106 cells) were extracted with detergent as described previously (13); protein concentrations in extracts were quantified by bicinchoninic acid assay (Pierce, Rockford, IL), and equal amounts of protein (up to 100 µg) were loaded per lane for gel electrophoresis. Samples were electrophoresed, electroblotted, and probed with antibodies to IL-2 receptors in analogy to our previous studies (13) with the following minor modifications. Samples were electrophoresed with 4% Tris-glycine gels (Invitrogen, Carlsbad, CA). IL-2Ralpha and IL-2Rgamma were probed with mouse anti-human antibodies clones 24204.11 and 38024.11, respectively (R&D Systems, Minneapolis, MN); stock solutions (500 µg/ml) of these antibodies were diluted 250-fold before use. Goat anti-mouse IgG peroxidase conjugate (Sigma, St. Louis, MO) was used as secondary antibody; the stock solution (7.5 mg/ml) of this antibody was diluted 50,000-fold before use. Antibody binding to receptors was visualized by chemiluminescence (SuperSignal chemiluminescent substrate; Pierce) as described previously (13).

Endocytosis of 125I-labeled IL-2. Radiolabeled IL-2 was used to determine whether biotin supply affects receptor-mediated endocytosis of IL-2. Two micrograms of recombinant human IL-2 (Biosource; lot. no. L122602B) were radiolabeled with 125I with a commercial kit according to the manufacturer's instructions (Iodo-Beads; Pierce). Bovine serum albumin was added after iodination (5 g/l final concentration) to prevent adsorption of IL-2 to plastic surfaces. Unbound 125I was removed by gel exclusion chromatography (D-Salt desalting column; Pierce). Specific radioactivity of the final product was ~11.5 GBq/mmol.

Endocytosis of 125I-IL-2 was measured in analogy to previous reports (38, 42). Briefly, 10 × 106 Jurkat cells were stimulated with 50 µg/l of PMA and 2 mg/l of PHA in a volume of 2.6 ml for 17 h to induce synthesis of IL-2R. Cells were washed with phosphate-buffered saline to remove secreted IL-2 and were incubated with 10 nM 125I-IL-2 at 4°C for 1 h in a volume of 1 ml; incubation was continued at 37°C for 10 min. Cells were pelleted by centrifugation (500 g for 3 min) and washed three times with 1 ml of phosphate-buffered saline (4°C). Cells were resuspended in a small volume of water, and 125I-IL-2 was quantified in a gamma counter.

Overexpression of IL-2Rgamma . Biotin deficiency causes decreased expression of IL-2Rgamma , leading to decreased endocytosis (and increased extracellular concentrations) of IL-2. Here we tested the hypothesis that overexpression of IL-2Rgamma abolishes the effects of biotin supply on extracellular levels of IL-2 by using an expression vector. With total RNA from Jurkat cells as template, cDNA was produced as described previously (45). PCR primers were designed to add SacI/KpnI restriction sites to the coding sequence of IL-2Rgamma (GenBank accession number D11086): 5'-ACT GAG CTC ATG TTG AAG CCA TCA TTA CCA-3' and 5'-AGA GGT ACC TCA GGT TTC AGG CTT TAG GGT-3' (Integrated DNA Technologies). The PCR product (spanning the full coding sequence of IL-2Rgamma ) was cloned with the AdvanTAge PCR cloning kit and the pT-Adv vector (Clontech, Palo Alto, CA). The IL-2Rgamma insert was sequenced five times at the DNA sequencing core facility at the University of Nebraska-Lincoln; the sequence was identical to the published sequence with the following exception. An A-to-G substitution was found in position 332 compared with the published sequence; the base triplets in both the published sequence (CTA) and our clone (CTG) encode the same amino acid (leucine), suggesting the existence of a silent variation in the IL-2Rgamma gene.

The SacI/KpnI fragment was subcloned into the pCMV-script expression vector (Stratagene, La Jolla, CA) according to the manufacturer's instructions; the resulting construct was denoted CMV-IL2Rgamma . Plasmid was extracted with a QIAprep Spin Miniprep kit (Qiagen).

Jurkat cells were transfected with CMV-IL2Rgamma by using SuperFect (Qiagen) to induce overexpression of IL-2Rgamma ; 24 h after transfection, cells were stimulated with PMA and PHA as described above to induce secretion of IL-2. Six hours after stimulation, cell-free supernatants were collected and assayed for IL-2 as described above.

Statistics. Homogeneity of variances among groups was tested with Bartlett's test (32). Variances were homogeneous; therefore, data were not transformed before further statistical testing. Significance of differences among groups was tested by one-way ANOVA. Fisher's protected least significant difference procedure was used for post hoc testing (32). StatView 5.0.1 (SAS Institute; Cary, NC) was used to perform all calculations. Differences were considered significant if P < 0.05. Data are expressed as means ± SD of separate experiments.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Propionyl-CoA carboxylase. Intracellular concentrations of biotin correlated with biotin concentrations in culture media, as judged by activities of PCC. When cells were cultured in biotin-deficient medium for 5 wk, activity of PCC was 0.7 ± 0.6 pmol bicarbonate fixed · 106 cells-1 · min-1 compared with 6.4 ± 0.9 pmol bicarbonate fixed · 106 cells-1 · min-1 in cells that were cultured in medium containing a physiological concentration of biotin; activity of PCC was 34 ± 4.3 pmol bicarbonate fixed · 106 cells-1 · min-1 when cells were cultured in medium containing a pharmacological concentration of biotin (P < 0.05; n = 3 experiments).

Secretion of IL-2 and expression of IL-2 gene. When cells were cultured in biotin-defined media for 5 wk, concentrations of free extracellular IL-2 (in kU IL-2 · l-1 · 106 cells-1 over 6 h) correlated negatively with biotin supply in response to stimulation with PMA and PHA: cells cultured in deficient medium, 4.1 ± 0.3; cells cultured in physiological medium, 3.0 ± 0.2; cells cultured in pharmacological medium, 1.8 ± 0.1 (P < 0.01; n = 5 experiments). This is consistent with previous observations (21, 43) and with the hypothesis that apparent secretion of IL-2 increases in response to biotin deficiency.

The increased apparent secretion of IL-2 by biotin-deficient cells was not paralleled by abundance of mRNA encoding IL-2. When cells were cultured in biotin-defined media for 5 wk, expression of the gene encoding IL-2 correlated positively with biotin supply; mRNA encoding IL-2 was barely detectable in biotin-deficient cells in response to stimulation with PMA and PHA for 6 h but was easily detectable in cells that were cultured in media containing physiological or pharmacological concentrations of biotin (Fig. 1A). Abundance of mRNA encoding IL-2 was quantified by gel densitometry and normalized by mRNA encoding G3PDH (control); abundance of mRNA (in arbitrary units of absorbance) was significantly greater in cells cultured in media containing 250 and 10,000 pM biotin compared with 25 pM biotin (P < 0.05) but was not significantly different in cells cultured with 250 pM biotin compared with 10,000 pM biotin (P > 0.05): cells cultured in deficient medium, 0.6 ± 0.2; cells cultured in physiological medium, 2.0 ± 0.9; cells cultured in pharmacological medium, 2.1 ± 0.3 (n = 3 experiments). Expression of the housekeeping gene G3PDH (control) was not significantly affected by biotin supply, as judged by gel densitometry (data not shown); Fig. 1A depicts a typical example from three experiments.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   Biotin supply correlates with transcriptional activity of the gene encoding interleukin (IL)-2. A: abundance of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (G3PDH; control) and IL-2 in Jurkat cells that were cultured in biotin-defined media for 5 wk, followed by stimulation with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutinin (PHA) for 6 h. mRNA was quantified by using reverse transcriptase polymerase chain reaction (PCR). B: luciferase activity in Jurkat cells that were cultured in biotin-defined media for 5 wk, followed by transfection with p(-321)IL2-Luc for 24 h. After transfection, cells were stimulated with PMA and PHA for 6 h and luciferase activity was quantified. Bars not sharing a superscript letter are significantly different (P < 0.05; n = 3 experiments).

Consistent with the observations made for mRNA abundance, luciferase activity in p(-321)IL2-Luc-transfected cells also correlated positively with biotin supply (Fig. 1B). These findings suggest that the increased apparent secretion of IL-2 by biotin-deficient cells was not caused by increased transcription of the IL-2 gene. Rather, the effects of biotin on the apparent secretion of IL-2 might be caused by processes such as receptor-mediated endocytosis.

Expression of genes encoding IL-2Ralpha and IL-2Rgamma . Expression of the IL-2Rgamma gene correlated positively with biotin concentrations in culture media. Both IL-2Rgamma mRNA and IL-2Rgamma protein were barely detectable in biotin-deficient cells and reached maximal levels in cells that were cultured in medium containing a pharmacological concentration of biotin (Fig. 2). In contrast, expression of IL-2Ralpha was not affected by biotin supply. Abundance of mRNA encoding the housekeeping gene G3PDH (control) was similar among treatment groups. These data are consistent with the hypothesis that biotin plays an important role in the expression of IL-2Rgamma in lymphoid cells.


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 2.   Biotin affects expression of the IL-2 receptor (R)gamma gene but not expression of genes encoding IL-2Ralpha and G3PDH (control). Jurkat cells were cultured in biotin-defined media for 5 wk and were stimulated with PMA and PHA for 21 h before sample collection. mRNA was quantified by reverse transcriptase PCR; receptor protein was quantified by Western blot analysis.

Does biotin-dependent expression of the IL-2Rgamma gene affect receptor-mediated endocytosis in Jurkat cells? Rates of IL-2 endocytosis correlated with cellular biotin supply and were about three times greater in cells cultured in medium containing a pharmacological concentration of biotin than in cells cultured in medium containing a deficient concentration of biotin (Fig. 3).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Biotin supply correlates with endocytosis of IL-2 in Jurkat cells. Cells were cultured in biotin-defined media for 5 wk and were stimulated with PMA and PHA for 21 h before sample collection. Secreted IL-2 was removed by washing, and rates of endocytosis were quantified by using 125I-labeled IL-2. Significantly different from cells cultured in medium containing 10,000 pM biotin: aP < 0.01, bP < 0.05 (n = 4 experiments).

Consistent with these findings, inhibition of IL-2Rgamma -mediated endocytosis by genistein abolished the effects of biotin on extracellular accumulation of IL-2. Here, cells were cultured in biotin-defined media for 5 wk, followed by stimulation with PMA and PHA for 6 h in the presence of 250 µM genistein. Apparent secretion of IL-2 (in kU IL-2 · l-1 · 106 cells-1 over 6 h) was not significantly different among treatment groups: 2.4 ± 0.5 (25 pM biotin), 1.9 ± 0.3 (250 pM biotin), and 1.8 ± 0.4 (10,000 pM biotin) (P > 0.05; n = 3 experiments). Together, these findings are consistent with the hypothesis that biotin-dependent expression of the IL-2Rgamma gene has a meaningful effect on receptor-mediated endocytosis of IL-2.

Luciferase activity correlated with biotin concentrations in cells that were transfected with pPBgamma 600 for 24 h: luciferase activity was significantly greater in cells cultured in medium containing 10,000 pM biotin than in cells cultured in medium containing 25 pM biotin (Fig. 4). Although the difference between the two treatments (25 vs. 10,000 pM) was moderate (~12%), increased luciferase activity in biotin-supplemented cells was consistent with observations regarding abundance of IL-2Rgamma mRNA and IL-2Rgamma protein (Fig. 2). The reporter gene studies suggest that biotin regulates expression of the IL-2Rgamma gene at the level of transcription.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Biotin affects expression of the IL-2Rgamma gene at the level of transcription. Luciferase activity was quantified in Jurkat cells that were cultured in biotin-defined media for 5 wk before transfection with pPBgamma 600 for 24 h. After transfection, cells were stimulated with PMA and PHA for 6 h and luciferase activity was quantified. * Significantly different from 10,000 pM biotin (n = 3 experiments).

Overexpression of IL-2Rgamma . Overexpression of IL-2Rgamma by transfection with CMV-IL-2Rgamma abolished the effects of biotin on extracellular accumulation (apparent secretion) of IL-2 in Jurkat cells. In plasmid CMV-IL-2Rgamma , expression of IL-2Rgamma is under control of the cytomegalovirus (CMV) promoter, leading to constitutive expression of IL-2Rgamma . Cells were cultured in biotin-defined media, transfected with CMV-IL-2Rgamma , and stimulated with PMA and PHA as described in Fig. 5. Transfection with CMV-IL-2Rgamma caused strong expression of IL-2Rgamma in all treatment groups, as judged by Western blot analysis with an antibody to IL-2Rgamma (Fig. 5A). Extracellular accumulation of IL-2 was not affected by biotin supply in CMV-IL-2Rgamma -transfected cells (Fig. 5B), unlike in nontransfected cells (see above). In fact, in contrast to nontransfected cells, we observed a moderate decrease of extracellular IL-2 in biotin-deficient cells. This was probably caused by the moderately greater abundance of IL-2Rgamma protein in CMV-IL-2Rgamma -transfected, biotin-deficient cells compared with the other treatment groups (Fig. 5A).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Overexpression of IL-2Rgamma in Jurkat cells abolishes effects of biotin on extracellular accumulation of IL-2. A: Jurkat cells were cultured in biotin-defined media for 5 wk before transfection with CMV-IL-2Rgamma for 24 h. After transfection, cells were stimulated with PMA and PHA for 6 h to induce secretion of IL-2 (B). Abundance of IL-2Rgamma protein was quantified by Western blot analysis with an antibody to IL-2Rgamma . B: extracellular IL-2 was quantified by ELISA. Bars not sharing a superscript letter are significantly different (P < 0.01; n = 3 experiments).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study provides evidence that 1) biotin supply affects expression of genes encoding IL-2 and IL-2Rgamma and 2) biotin-induced expression of IL-2Rgamma causes increased receptor-mediated endocytosis (i.e., decreased apparent secretion) of IL-2 in lymphoid cells. These effects of biotin are probably not specific for a given lymphoid cell line such as the Jurkat cells used here; biotin supplementation also caused decreased apparent secretion of IL-2 in previous studies in freshly isolated human lymphocytes (43).

The present study and previous studies (21, 43) are consistent with the hypothesis that biotin supplementation will benefit the immune system by the following two mechanisms. 1) Biotin-supplemented cells produce more IL-2 than biotin-deficient cells (as judged by abundance of mRNA), potentially leading to increased growth and differentiation of T cells, natural killer cells and B cells, and some myeloid cells (17). Consistent with this hypothesis, proliferation rates increased transiently in biotin-supplemented Jurkat cells compared with controls that were cultured in medium containing a physiological concentration of biotin (21). 2) Synthesis of IL-2Rgamma correlates with biotin concentrations in human lymphoid cells. Effects of biotin on the expression of the IL-2Rgamma gene are likely to be beneficial because IL-2Rgamma plays a role not only in endocytosis of IL-2 (leading to degradation of IL-2) but also in triggering intracellular signaling pathways leading to growth and differentiation of immune cells in response to IL-2 (17, 31, 37). Only those IL-2R (heteromers) that contain IL-2Rgamma can mediate IL-2-signaling: high-affinity and intermediate-affinity receptors (37, 39). This is consistent with an essential role for IL-2Rgamma in signal transduction and in the human immune response. Mutations of the gene encoding IL-2Rgamma (as seen in human X-linked severe combined immunodeficiency) are fatal if not cured by bone marrow transplantation, consistent with a pivotal role for IL-2Rgamma in the immune system (37). IL-2Rgamma is also assembled into receptor heteromers for IL-4, -7, -9, and -15, underlining the physiological importance of this receptor (17, 37). The present study offers an explanation for the following paradox observed in previous studies: biotin supplementation causes decreased apparent secretion of IL-2 (21, 43) despite the fact that biotin plays an essential role in the immune response (3, 7, 18, 28-30). The possibility that biotin increases the binding affinity of IL-2 to its receptor(s) cannot be excluded on the basis of the data presented here.

The observed effects on receptor-mediated endocytosis of IL-2 are caused specifically by biotin rather than by a global increase in transcriptional activity in response to cellular nutrient supply, on the basis of the following lines of reasoning. 1) Biotin did not affect expression of genes encoding IL-2Ralpha and G3PDH, consistent with the hypothesis that biotin does not globally increase transcriptional activity. 2) Transcriptional activity of genes encoding IL-2 and IL-2Rgamma did not increase in response to supplementation with the vitamin riboflavin compared with riboflavin-deficient cells (unpublished observation). This is consistent with the hypothesis that transcription of the genes encoding IL-2 and IL-2Rgamma responds specifically to biotin.

There is precedence for the effects of biotin on gene expression. For example, biotin affects transcription of genes encoding glucokinase, phosphoenolpyruvate carboxykinase, and ornithine transcarbamylase (4, 6, 8-10, 20, 34). The following models might explain how biotin affects gene expression. 1) An intermediate of biotin metabolism, biotinyl-AMP, activates soluble guanylate cyclase, leading to increased production of cGMP (33). cGMP stimulates protein kinase G (33), leading to phosphorylation of enzymes and other proteins that regulate processes such as transcription. 2) Histones (DNA-binding proteins) in human lymphocytes are modified by covalent attachment of biotin (35). Various covalent modifications of histones may lead to increased transcriptional activity of genes (40) such as IL-2 (5). Consistent with these observations, biotinylation of histones correlates with increased proliferation of human lymphocytes (35), a process that depends on IL-2. 3) Biotin may bind to transcription factors that affect expression of genes encoding IL-2 and IL-2Rgamma . The identity of these transcription factors is currently unknown.

In the present study PCC activity was used as a marker for intracellular concentrations of biotin. Previous studies provided evidence that PCC activity is an early and sensitive marker for biotin status in rats (24); biotinylation of PCC (abundance of holo-PCC) correlates with concentrations of intracellular free biotin in rat livers (19). Likewise, biotinylation of PCC and other carboxylases in Jurkat cells correlates with biotin supply in culture media (21). Together, these studies are consistent with the hypothesis that activities of PCC in mammalian cells correlate with intracellular concentrations of free biotin, biotinylation of carboxylases, and cellular biotin supply.

The effects of nutrient status on immune function were investigated in numerous previous studies. These studies frequently used secretion of cytokines by immune cells as a marker to model effects of nutrient deficiency or supplementation on immune function. The present study suggests that the approach of quantifying apparent secretion of cytokines without quantifying cytokine receptors may be too simplistic. Previous studies that relied solely on the analysis of extracellular cytokine levels may require careful reevaluation.


    ACKNOWLEDGEMENTS

We thank Drs. H. Asao (Tohoku University School of Medicine), L. P. Freedman (Memorial Sloan-Kettering Cancer Center), and B. R. White (University of Nebraska-Lincoln) for generously providing plasmids for these studies.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-60447 and U.S. Department of Agriculture/National Research Initiative Competitive Grants Program Project Award 2001-35200-10187. This article is a contribution by the University of Nebraska Agricultural Research Division, Lincoln, NE 68583 (Journal Series No. 13792).

Address for reprint requests and other correspondence: J. Zempleni, Dept. of Nutritional Sciences and Dietetics, Univ. of Nebraska at Lincoln, 316 Ruth Leverton Hall, Lincoln, NE 68583-0806 (E-mail: jzempleni2{at}unl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 16, 2002;10.1152/ajpcell.00365.2002

Received 23 September 2002; accepted in final form 7 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alroy, I, Towers TL, and Freedman LP. Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. Mol Cell Biol 15: 5789-5799, 1995[Abstract].

2.   Arcari, P, Martinelli R, and Salvatore F. The complete sequence of a full length cDNA for liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species. Nucleic Acids Res 12: 9179-9189, 1984[Abstract].

3.   Báez-Saldaña, A, Díaz G, Espinoza B, and Ortega E. Biotin deficiency induces changes in subpopulations of spleen lymphocytes in mice. Am J Clin Nutr 67: 431-437, 1998[Abstract].

4.   Borboni, P, Magnaterra R, Rabini RA, Staffolani R, Porzio O, Sesti G, Fusco A, Mazzanti L, Lauro R, and Marlier LN. Effect of biotin on glucokinase activity, mRNA expression and insulin release in cultured beta-cells. Acta Diabetol 33: 154-158, 1996[ISI][Medline].

5.   Butscher, WG, Haggerty CM, Chaudhry S, and Gardner K. Targeting of p300 to the interleukin-2 promoter via CREB-Rel cross-talk during mitogen and oncogenic molecular signaling in activated T cells. J Biol Chem 276: 27647-27656, 2001[Abstract/Free Full Text].

6.   Chauhan, J, and Dakshinamurti K. Transcriptional regulation of the glucokinase gene by biotin in starved rats. J Biol Chem 266: 10035-10038, 1991[Abstract/Free Full Text].

7.   Cowan, MJ, Wara DW, Packman S, Yoshino M, Sweetman L, and Nyhan W. Multiple biotin-dependent carboxylase deficiencies associated with defects in T-cell and B-cell immunity. Lancet 2: 115-118, 1979[ISI][Medline].

8.   Dakshinamurti, K, and Chauhan J. Biotin-binding proteins. In: Vitamin Receptors: Vitamins as Ligands in Cell Communication, edited by Dakshinamurti K.. Cambridge, UK: Cambridge Univ. Press, 1994.

9.   Dakshinamurti, K, and Cheah-Tan C. Liver glucokinase of the biotin deficient rat. Can J Biochem 46: 75-80, 1968[ISI][Medline].

10.   Dakshinamurti, K, and Litvak S. Biotin and protein synthesis in rat liver. J Biol Chem 245: 5600-5605, 1970[Abstract/Free Full Text].

11.   Fujita, T, Shibuya H, Ohashi T, Yamanishi K, and Taniguchi T. Regulation of human interleukin-2 gene: functional DNA sequences in the 5'-flanking region of the gene expression in activated T lymphocytes. Cell 46: 401-407, 1986[ISI][Medline].

12.   Gnarra, JR, Otani H, Wang MG, McBride OW, Sharon M, and Leonard WJ. Human interleukin 2 receptor beta -chain gene: chromosomal localization and identification of 5' regulatory sequences. Proc Natl Acad Sci USA 87: 3440-3444, 1990[Abstract].

13.   Griffin, JB, Stanley JS, and Zempleni J. Synthesis of a rabbit polyclonal antibody to the human sodium-dependent multivitamin transporter. Int J Vitam Nutr Res 72: 195-198, 2002[Medline].

14.   Hatakeyama, M, Tsudo M, Minamoto S, Kono T, Doi T, Miyata T, Miyasaka M, and Taniguchi T. Interleukin-2 receptor beta  chain gene: generation of three receptor forms by cloned human alpha  and beta  chain cDNAs. Science 244: 551-556, 1989[ISI][Medline].

15.   Hemar, A, and Dautry-Varsat A. Cyclosporin A inhibits the interleukin 2 receptor alpha  chain gene transcription but not its cell surface expression: the alpha  chain stability can explain this discrepancy. Eur J Immunol 20: 2629-2635, 1990[ISI][Medline].

16.   Hentsch, B, Mouzaki A, Pfeuffer I, Rungger D, and Serfling E. The weak, fine-tuned binding of ubiquitous transcription factors to the IL-2 enhancer contributes to its T cell-restricted activity. Nucleic Acids Res 20: 2657-2665, 1992[Abstract].

17.   Klein, J, and Horejsi V. Immunology. Oxford, UK: Blackwell Science, 1997.

18.   Kumar, M, and Axelrod AE. Cellular antibody synthesis in thiamin, riboflavin, biotin and folic acid-deficient rats. Proc Soc Exp Biol Med 157: 421-423, 1978.

19.   Lewis, B, Rathman S, and McMahon R. Dietary biotin intake modulates the pool of free and protein-bound biotin in rat liver. J Nutr 131: 2310-2315, 2001[Abstract/Free Full Text].

20.   Maeda, Y, Kawata S, Inui Y, Fukuda K, Igura T, and Matsuzawa Y. Biotin deficiency decreases ornithine transcarbamylase activity and mRNA in rat liver. J Nutr 126: 61-66, 1996[ISI][Medline].

21.   Manthey, KC, Griffin JB, and Zempleni J. Biotin supply affects expression of biotin transporters, biotinylation of carboxylases, and metabolism of interleukin-2 in Jurkat cells. J Nutr 132: 887-892, 2002[Abstract/Free Full Text].

22.   Mock, DM. Determinations of biotin in biological fluids. Methods Enzymol 279: 265-275, 1997[ISI][Medline].

23.   Mock, DM, Lankford GL, and Mock NI. Biotin accounts for only half of the total avidin-binding substances in human serum. J Nutr 125: 941-946, 1995[ISI][Medline].

24.   Mock, DM, and Mock NI. Lymphocyte propionyl-CoA carboxylase is an early and sensitive indicator of biotin deficiency in rats, but urinary excretion of 3-hydroxypropionic acid is not. J Nutr 132: 1945-1950, 2002[Abstract/Free Full Text].

25.   Morelon, E, and Dautry-Varsat A. Endocytosis of the common cytokine receptor gamma c chain. J Biol Chem 273: 22044-22051, 1998[Abstract/Free Full Text].

26.   National Research Council. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B-6, folate, vitamin B-12, pantothenic acid, biotin, and choline. In: Recommended Dietary Allowances, edited by Food and Nutrition Board. Washington, DC: National Academy, 1998.

27.   Ohbo, K, Takasawa N, Ishii N, Tanaka N, Nakamura M, and Sugamura K. Functional analysis of the human interleukin-2 receptor gamma  chain promoter. J Biol Chem 270: 7479-7486, 1995[Abstract/Free Full Text].

28.   Petrelli, F, Moretti P, and Campanati G. Studies on the relationships between biotin and the behaviour of B and T lymphocytes in the guinea pig. Experientia 37: 1204-1206, 1981[ISI][Medline].

29.   Pruzansky, J, and Axelrod AE. Antibody production to diphtheria toxoid in vitamin deficiency states. Proc Soc Exp Biol Med 89: 323-325, 1955.

30.   Rabin, BS. Inhibition of experimentally induced autoimmunity in rats by biotin deficiency. J Nutr 113: 2316-2322, 1983[ISI][Medline].

31.   Russell, SM, Johnston JA, Noguchi M, Kawamura M, Bacon CM, Friedman M, Berg M, McVicar DW, Witthuhn BA, Silvennoinen O, Goldman AS, Schmalstieg FC, Ihle JN, O'Shea JJ, and Leonard WJ. Interactions of IL-2Rbeta and gamma c chains with Jak1 and Jak3: implications for XSCID and XCID. Science 266: 1042-1045, 1994[ISI][Medline].

32.   SAS Institute. StatView Reference. Cary, NC: SAS, 1999.

33.   Solorzano-Vargas, RS, Pacheco-Alvarez D, and Leon-Del-Rio A. Holocarboxylase synthetase is an obligate participant in biotin-mediated regulation of its own expression and of biotin-dependent carboxylases mRNA levels in human cells. Proc Natl Acad Sci USA 99: 5325-5330, 2002[Abstract/Free Full Text].

34.   Spence, JT, and Koudelka AP. Effects of biotin upon the intracellular level of cGMP and the activity of glucokinase in cultured rat hepatocytes. J Biol Chem 259: 6393-6396, 1984[Abstract/Free Full Text].

35.   Stanley, JS, Griffin JB, and Zempleni J. Biotinylation of histones in human cells: effects of cell proliferation. Eur J Biochem 268: 5424-5429, 2001[Abstract/Free Full Text].

36.   Subtil, A, and Dautry-Varsat A. Several weak signals in the cytosolic and transmembrane domains of the interleukin-2-receptor beta  chain allow for its efficient endocytosis. Eur J Biochem 253: 525-530, 1998[Abstract].

37.   Sugamura, K, Asao H, Kondo M, Tanaka N, Ishii N, Ohbo K, Nakamura M, and Takeshita T. The interleukin-2 receptor gamma  chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu Rev Immunol 14: 179-205, 1996[ISI][Medline].

38.   Takeshita, T, Asao H, Ohtani K, Ishii N, Kumaki S, Tanaka N, Munakata H, Nakamura M, and Sugamura K. Cloning of the gamma  chain of the human IL-2 receptor. Science 257: 379-382, 1992[ISI][Medline].

39.   Taniguchi, T, and Minami Y. The IL-2/IL-2 receptor system: a current overview. Cell 73: 5-8, 1993[ISI][Medline].

40.   Wolffe, A. Chromatin. San Diego: Academic, 1998.

41.   Yu, A, and Malek TR. The proteasome regulates receptor-mediated endocytosis of interleukin-2. J Biol Chem 276: 381-385, 2001[Abstract/Free Full Text].

42.   Yu, A, Olosz F, Choi CY, and Malek TR. Efficient internalization of IL-2 depends on the distal portion of the cytoplasmic tail of the IL-2R common gamma -chain and a lymphoid cell environment. J Immunol 165: 2556-2562, 2000[Abstract/Free Full Text].

43.   Zempleni, J, Helm RM, and Mock DM. In vivo biotin supplementation at a pharmacologic dose decreases proliferation rates of human peripheral blood mononuclear cells and cytokine release. J Nutr 131: 1479-1484, 2001[Abstract/Free Full Text].

44.   Zempleni, J, and Mock DM. Uptake and metabolism of biotin by human peripheral blood mononuclear cells. Am J Physiol Cell Physiol 275: C382-C388, 1998[Abstract/Free Full Text].

45.   Zempleni, J, Stanley JS, and Mock DM. Proliferation of peripheral blood mononuclear cells causes increased expression of the sodium-dependent multivitamin transporter gene and increased uptake of pantothenic acid. J Nutr Biochem 12: 465-473, 2001[ISI][Medline].

46.   Zempleni, J, Trusty TA, and Mock DM. Lipoic acid reduces the activities of biotin-dependent carboxylases in rat liver. J Nutr 127: 1776-1781, 1997[Abstract/Free Full Text].


Am J Physiol Cell Physiol 284(2):C415-C421
0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society