HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Menné, C. Articles by Geisler, C. Search for Related Content PubMed PubMed Citation Articles by Menné, C. Articles by Geisler, C.

Ceramide-Induced TCR Up-Regulation¹

Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The TCR is a constitutively recycling receptor meaning that a constant fraction of TCR from the plasma membrane is transported inside the cell at the same time as a constant fraction of TCR from the intracellular pool is transported to the plasma membrane. TCR recycling is affected by protein kinase C activity. Thus, an increase in protein kinase C activity affects TCR recycling kinetics leading to a new TCR equilibrium with a reduced level of TCR expressed at the T cell surface. Down-regulation of TCR expression compromises T cell activation. Conversely, TCR up-regulation is expected to increase T cell responsiveness. The purpose of this study was to identify and characterize potential pathways for TCR up-regulation. We found that ceramide affected TCR recycling dynamics and induced TCR up-regulation in a concentration- and time-dependent manner. Experiments applying phosphatase inhibitors indicated that ceramide-induced TCR up-regulation was most probably mediated by serine/threonine protein phosphatase 2A. Analyses of T cell variants demonstrated that TCR up-regulation was dependent on the presence of an intact CD3 L-based motif and thus acted on TCR engaged in the recycling pathway. Finally, we showed that TCR up-regulation probably plays a physiological role by increasing T cell responsiveness. Thus, by affecting the TCR recycling kinetics, T cells have the potential both to up- and down-regulate TCR expression and thereby adjust T cell responsiveness.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Regulation of TCR cell surface expression levels is most probably an important mechanism by which T cell responsiveness is controlled. Accordingly, several studies have demonstrated that the capacity to reach T cell activation thresholds following ligand-mediated TCR stimulation is dependent on the number of TCR expressed at the cell surface (1, 2, 3). Likewise, regulation of TCR expression seems to play an important role during positive and negative selection of thymocytes (4). It is well-known that the TCR is a constitutively recycling receptor (3, 5, 6, 7). For the recycling pool of TCR in steady state the following equation is valid:

(1)

where k_in is the rate constant in (rate of TCR endocytosed per min), [TCR]_s the number of TCR expressed at the cell surface, k_out the rate constant out (rate of TCR exocytosed per min), and [TCR]_i the number of intracellular TCR. By altering k_in, k_out, or both, T cells have the potential to regulate TCR expression levels and thereby probably T cell responsiveness. The molecular mechanisms that determine k_in are well-characterized. Thus, k_in is determined by phosphorylation of serine 126 of the TCR subunit CD3 (5, 6, 8, 9). Following protein kinase C (PKC)³-mediated phosphorylation of serine 126, the CD3 leucine-based receptor-sorting motif (L-based motif) becomes accessible for clathrin-coated vesicle adaptor protein-2 binding (2). Binding of adaptor protein-2 to the CD3 L-based motif results in increased internalization and down-regulation of the TCR from the T cell surface. Following PKC-mediated internalization, the TCR is not degraded but retained in intracellular vesicles until recycled back to the cell surface (3). In contrast, the molecular mechanisms determining k_out are unknown. A previous study has suggested that the serine/threonine protein phosphatase 2A (PP2A) might play a role in TCR recycling. Thus, inhibition of PP2A leads to TCR down-regulation, which could be explained by an increase of k_in (9). However, the possibility existed that inhibition of PP2A activity resulted in TCR down-regulation by a reduction of k_out. If this is the case it would be expected that an increase in PP2A activity leads to TCR up-regulation by increasing k_out.

Several pathways with the potential to increase PP2A activity exist in T cells during their encounter with APC. For instance, triggering of the TCR (10), CD5 (11), CD28 (12), and cytokine receptors for IL-1ß (13) and TNF- (14, 15) activates sphingomyelinase. Sphingomyelinase in turn hydrolyzes sphingomyelin to generate the lipid second messenger ceramide that is a potent activator of ceramide-activated protein phosphatase belonging to the PP2A family (16, 17, 18). In addition, activated macrophages directly release sphingomyelinase (19). The list of biological effects attributable to ceramide is continually expanding and includes roles in the regulation of cell proliferation and differentiation, inflammation, and apoptosis, dependent on cell type and concentrations of ceramide (20, 21).

The aim of this study was to determine mechanisms by which T cells are able to up-regulate TCR expression levels. We found that ceramide induced TCR up-regulation in freshly isolated human T cells and T cell lines in a concentration- and time-dependent manner by increasing k_out. Experiments applying phosphatase inhibitors indicated that ceramide-induced TCR up-regulation was most probably mediated by PP2A. Analyses of T cell variants demonstrated that TCR up-regulation was dependent on the presence of an intact CD3 L-based motif. However, studies of chimeric CD16/CD3 molecules showed that neither the CD3 L-based motif nor the chain was sufficient to mediate receptor exocytosis. Finally, we showed that TCR up-regulation might play a physiological role by increasing T cell responsiveness.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and reagents

PBMC were isolated from healthy donors by density centrifugation using Lymphoprep as described by the manufacturer (Nycomed Pharma AS, Oslo, Norway). J45 is a CD45-negative variant of the Jurkat T cell line (22). CD3-negative Jurkat variants were transfected with cDNA coding for either wild-type CD3 (CD3-WT), CD3-LLAA in which alanines substituted for leucine 131 and leucine 132, CD3-P133 in which the cytoplasmic tail of CD3 is truncated at proline 133, or the chimeric CD16/CD3-tP133 molecule consisting of the extracellular and transmembrane part of FcRIIIA- (CD16) and the cytoplasmic part of CD3 from glutamine 117 to leucine 132 as previously described (8, 23, 24). C₂-ceramide (C2) and the Vybrant apoptosis assay kit were obtained from Molecular Probes (Leiden, The Netherlands). Dihydro-C₂-ceramide (DHC2) and calyculin A were obtained from Biomol (Plymouth Meeting, PA). Okadaic acid and tautomycin were obtained from Calbiochem (San Diego, CA). Fluorochrome-conjugated anti-CD2, -CD3, -CD4, -CD5, -CD8, -CD16, -CD25, and CD69 mAbs were obtained from Leinco Technologies (Ballwin, MO) and PharMingen (Becton Dickinson, Mountain View, CA). The phorbol-ester phorbol 12,13-dibutyrate (PDB) was obtained from Sigma (St. Louis, MO).

TCR up- and down-regulation

Cells were resuspended at a concentration of 4 x 10⁵/ml in RPMI 1640 medium (Sigma) supplemented with either 2% (v/v) pooled human serum type AB (PBMC) or 10% (v/v) FCS (Jurkat cells) and incubated at 37°C in 5% CO₂ in 96-well microplates from Nunc (Roskilde, Denmark). Cells were incubated in triplicate or quadruplicate with the indicated reagents, transferred to ice-cold PBS containing 2% FCS and 0.1% NaN₃, and washed. The cells were directly stained with fluorochrome-conjugated mAb and analyzed by flow cytometry using a FACScalibur flow cytometer (Becton Dickinson). Mean fluorescence intensity (MFI) was recorded and used in the calculation of percent mAb binding: ((MFI of treated cells)/(MFI of untreated cells)) x 100%. All experiments were repeated at least three times.

Apoptosis assay

Cells were resuspended at a concentration of 4 x 10⁵/ml in RPMI 1640 medium and incubated with 40 µM C2 for the indicated time at 37°C in 5% CO₂ in 96-well microplates. The cells were subsequently washed in ice-cold PBS, resuspended in 1x annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4), incubated with Alexa 488-conjugated annexin V at room temperature for 15 min, and then analyzed by flow cytometry. The percent of apoptotic cells in a sample was calculated as ((cells positive for annexin V)/(total number of cells)) x 100%.

TCR internalization and recycling

To determine the rate constant in of the TCR, J45 cells were incubated at a cell density of 1.8 x 10⁶ cells/ml medium at 4°C with PE-conjugated anti-CD3 mAb for 30 min. The cells were washed and subsequently resuspended in 37°C medium containing either 25 µM C2 or 100 nM PDB. At the time indicated, aliquots of cell suspension were washed in ice-cold PBS containing 2% FCS and 0.1% NaN₃ and immediately treated with 300 µl 0.5 M NaCl, 0.5 M acetic acid, pH 2.2, for 10 s. The acid-resistant fluorescence of the cells (representing internalized anti-CD3 mAb) was measured in the FACScalibur. The percentage of internalized anti-CD3 mAb to cell surface-bound anti-CD3 mAb was subsequently calculated using the equation: ((HAR - CAR)/CT) x 100%, where HAR is the MFI of acid-treated cells incubated at 37°C, CAR is the MFI of acid-treated cells incubated at 4°C, and CT is the MFI of untreated cells incubated at 4°C.

For analysis of receptor recycling, cells were incubated for 60 min with PDB, washed three times to remove PDB, and subsequently incubated in medium without PDB at 37°C to allow receptor recycling. At different time points, aliquots of the cells were stained with PE-conjugated anti-CD3 or anti-CD16 mAb and analyzed in a FACScalibur flow cytometer. MFI was recorded and used in the calculation of percent anti-CD3 or anti-CD16 mAb binding: ((MFI of PDB treated cells)/(MFI of untreated cells)) x 100%. For each construct, three independent transfectants were analyzed.

CD69 and CD25 up-regulation

To determine CD69 and CD25 up-regulation, cells were resuspended in triplicate at a concentration of 4 x 10⁵ cells/ml medium and pretreated with 25 µM C2 for 30 min at 37°C in 5% CO₂ or left untreated. Subsequently, the cells were washed three times to remove C2 and incubated in ice-cold medium containing 100 ng/ml anti-human TCR mAb F101.01 (25) for 30 min on ice. The cells were washed three times to remove unbound mAb and resuspended in 37°C medium. Following incubation for 22 h at 37°C in 5% CO₂ the cells were transferred to ice-cold PBS containing 2% FCS and 0.1% NaN₃, directly stained with PE-conjugated anti-CD69 or anti-CD25 mAb, and analyzed by flow cytometry. MFI was recorded and used in the calculation of percent mAb binding.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ceramide induces TCR up-regulation

To examine the effect of ceramide on TCR expression, freshly isolated PBMC from healthy donors were incubated with various concentrations of the cell-permeable C2 for 15 min. The cells were washed, stained with fluorochrome-conjugated mAbs, and subsequently analyzed by flow cytometry. C2 induced a 15–20% up-regulation of the TCR expression levels. The effect of C2 appeared to be selective for the TCR as C2 did not induce up-regulation of CD2, CD4, CD5, or CD8 (Fig. 1A and data not shown). To investigate whether C2 influenced TCR expression equally on CD4⁺ and CD8⁺ T cells, cells were incubated with C2 or the presumably inactive DHC2 for 15 min and double-stained with either anti-CD3 plus anti-CD4 mAb or anti-CD3 plus anti-CD8 mAb. C2 induced comparable TCR up-regulation between 15 and 20% in both CD4⁺ and CD8⁺ T cells (Fig. 1B). The increase in TCR staining observed after C2 treatment was caused by a homogeneously TCR up-regulation in both CD4⁺ and CD8⁺ populations (Fig. 1, C and D). In contrast, DHC2 only induced an 5% up-regulation in TCR expression at the highest concentration tested (Fig. 1B). The small up-regulatory effect of DHC2 on TCR expression might be due to the reported conversion of DHC2 into C2 (26).

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Ceramide induces TCR up-regulation. A, PBMC were incubated with C2 for 15 min at 37°C. The cells were washed, stained with PE-conjugated anti-CD3 mAb and either FITC-conjugated anti-CD2 or anti-CD5 mAb, and analyzed by flow cytometry. Receptor up-regulation was determined by comparing MFI of C2-treated cells with MFI of untreated cells. The experiments were performed in triplicates, and the results are given as mean ± SD. Comparable results were obtained in four independent experiments using PBMC from different donors. B, PBMC were incubated with C2 or DHC2 for 15 min at 37°C. The cells were washed, stained with PE-conjugated anti-CD3 mAb and either FITC-conjugated anti-CD4 or anti-CD8 mAb, and analyzed by flow cytometry. TCR up-regulation was determined by comparing anti-CD3 MFI of C2/DHC2-treated cells with MFI of untreated cells. The experiments were performed in triplicates, and the results are given as mean ± SD. Comparable results were obtained in at least five independent experiments with different donors. (CD3(4+), CD3 expression on CD4-positive cells; CD3(8+), CD3 expression on CD8-positive cells). C and D, FACS profiles of CD4+ (C) and CD8+ (D) cells untreated (black) or treated with C2 (40 µM) (stippled line) for 15 min. The abscissa gives the anti-CD3 fluorescence intensity in a logarithmic scale. The ordinate gives the relative cell number. The distance given by the bar represents a 100% up-regulation.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Ceramide induces a rapid, long-lasting, and reversible TCR up-regulation. A, PBMC were incubated with 25 µM C2 for the time indicated at 37°C. The cells were washed, stained with PE-conjugated anti-CD3 mAb and either FITC-conjugated anti-CD4 or anti-CD8 mAb, and analyzed by flow cytometry. TCR up-regulation was determined by comparing anti-CD3 MFI of C2-treated cells with MFI of untreated cells. The results are given as mean of triplicate experiments. Comparable results were obtained in three independent experiments with different donors. B, PBMC were incubated with 25 µM C2 for 15 min at 37°C. The cells were washed to remove ceramide and incubated at 37°C. At the time indicated, aliquots of the cells were transferred to ice-cold PBS, stained with PE-conjugated anti-CD3 mAb and either FITC-conjugated anti-CD4 or anti-CD8 mAb, and analyzed by flow cytometry. TCR up-regulation was determined by comparing anti-CD3 MFI of C2-treated cells with MFI of untreated cells. The results are given as mean of triplicate experiments. Comparable results were obtained in three independent experiments with different donors. C, T cells were incubated with 40 µM C2 for the time indicated at 37°C. The cells were washed and stained with Alexa 488-conjugated annexin V and analyzed by flow cytometry. Comparable results were obtained in three independent experiments.

Taken together, these experiments demonstrated that C2 induces a rapid, long-lasting, and reversible up-regulation of TCR expression levels in freshly isolated CD4⁺ and CD8⁺ T cells.

Ceramide-induced TCR up-regulation is mediated via PP2A

Ceramide is a known activator of ceramide-activated protein phosphatases that belong to the serine/threonine protein phosphatase family PP2A (17, 18). To investigate whether the up-regulatory effect of C2 on TCR expression was mediated by activation of serine/threonine protein phosphatases, cells were preincubated with different protein phosphatase inhibitors for 1 h and subsequently treated with C2 for 15 min. As shown in Fig. 3, A and B, the potent PP2A inhibitors calyculin A and okadaic acid completely inhibited C2-induced TCR up-regulation at concentrations of 120 and 500 nM, respectively. In contrast, the protein phosphatase 1 inhibitor tautomycin did not affect the C2-induced TCR up-regulation even at the highest concentrations tested (Fig. 3C). These experiments suggested that C2 induces TCR up-regulation by activation of serine/threonine protein phosphatases belonging to the PP2A group.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Ceramide-induced TCR up-regulation is mediated via PP2A. PBMC were preincubated with (A) calyculin A (Cal A), (B) okadaic acid (OA), or (C) tautomycin (Tau) for 1 h at 37°C. The cells were then incubated with 25 µM C2 for 15 min at 37°C, washed, stained with PE-conjugated anti-CD3 mAb and either FITC-conjugated anti-CD4 or anti-CD8 mAb, and analyzed by flow cytometry. TCR up-regulation was determined by comparing anti-CD3 MFI of C2-treated cells with MFI of non-C2-treated cells. The results are given as mean of triplicate experiments. Comparable results were obtained in three independent experiments using PBMC from different donors.

TCR up-regulation can be mediated either by an increase of k_out, a decrease of k_in, or a combination of the two. To determine how ceramide-induced activation of PP2A influenced the rate constants, k_in was measured in untreated J45 cells or J45 cells treated with either ceramide or the PKC activator PDB as a control. We used the CD45-negative cell line J45 in these experiments as we have previously shown that anti-TCR Abs induce only minimal TCR internalization in this cell line (9). As shown in Fig. 4, ceramide treatment did not affect k_in, whereas PDB treatment caused an increase of k_in as previously described (5, 9). As ceramide mediated TCR up-regulation without affecting k_in, we could conclude from these experiments that TCR up-regulation following ceramide-induced activation of PP2A was mediated by an increase of k_out.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Ceramide-induced TCR up-regulation is mediated by an increase of kout. J45 cells were incubated with PE-conjugated anti-CD3 mAb for 30 min at 4°C. The cells were washed and subsequently resuspended in 37°C medium containing either 25 µM C2 or 100 nM PDB. At the time indicated, aliquots of the cells were analyzed for acid-resistant fluorescence representing internalized anti-CD3 mAb by flow cytometry. The percentage of internalized anti-CD3 mAb to cell surface-bound anti-CD3 mAb was subsequently calculated as described in Materials and Methods. Comparable results were obtained in four independent experiments.

In theory, during TCR up-regulation TCR can be recruited from either the recycling pool of TCR, from newly synthezised TCR, or from a combination of the two. We have previously shown that PKC-mediated TCR internalization and TCR recycling is dependent on the L-based motif in CD3. Thus, mutation of leucine 131 and 132 in the cytoplasmic tail of CD3 to alanine (LLAA) completely abolishes PKC-mediated TCR down-regulation, and no pool of recycling TCR is found in CD3-LLAA transfectants (2, 3, 8, 9). To analyze whether ceramide-induced TCR up-regulation was dependent on the presence of a recycling pool of TCR, the CD3-WT and CD3-LLAA transfectants were studied. CD3-WT and CD3-LLAA cells express comparable levels of TCR at their cell surface. The cells were incubated with various concentrations of C2 for 15 min, washed, and subsequently analyzed for TCR expression by flow cytometry. C2 at concentrations of 25 µM and above induced 15–20% TCR up-regulation in CD3-WT cells. In contrast, in CD3-LLAA cells with a disrupted CD3 L-based motif TCR expression was left almost unaffected by C2 (Fig. 5). These experiments demonstrated that PP2A-induced TCR up-regulation was independent of newly synthesized TCR but dependent on an intact CD3 L-based motif and thus most probably on the presence of a recycling pool of TCR.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. TCR up-regulation is dependent on the CD3 L-based motif. CD3-WT and CD3-LLAA cells were incubated with C2 for 15 min at 37°C. The cells were washed, stained with PE-conjugated anti-CD3 mAb, and analyzed by flow cytometry. TCR up-regulation was determined by comparing MFI of C2-treated cells with MFI of untreated cells. The results are given as mean of triplicate experiments. Comparable results were obtained in three independent experiments.

Neither the CD3 leucine-based receptor-sorting motif nor the -chain is sufficient to mediate receptor exocytosis

It is still unknown whether TCR recycling occurs by default or whether it is mediated by recycling signals. However, for some receptors, such as ß₂ integrins, tyrosine-based signals are involved in sorting internalized receptors to recycling vesicles (27). To determine whether the CD3 L-based motif or motifs in the -chain were responsible for TCR exocytosis following TCR internalization, we next studied recycling of the chimeric CD16/CD3-tP133 molecule compared with the TCR/CD3-tP133 expressing identical CD3 cytoplasmic tails. CD16/CD3-tP133 contains an intact CD3 L-based motif and is expressed at the cell surface in association with the homodimer. Following PKC activation, the CD16/CD3- complex is internalized like the TCR (24). Cells were treated for 60 min with PDB, washed three times to remove PDB, and subsequently incubated in medium without PDB at 37°C to allow receptor recycling. At different time points, aliquots of the cells were analyzed for CD16/CD3 and TCR expression. PDB induced comparable down-regulation of CD16/CD3-tP133 and the TCR to 40% compared with nontreated cells (Fig. 6). However, after withdrawal of PDB the TCR rapidly recycled back to the plasma membrane and reached 100% expression in 60 min, whereas CD16/CD3-tP133 did not recycle to any significant extent (Fig. 6).

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Neither the CD3 L-based motif nor the -chain is sufficient to mediate receptor exocytosis. A, Schematic presentation of the TCR/CD3-tP133 and CD16/CD3-tP133- complexes. B, TCR/CD3-tP133 and CD16/CD3-tP133 cells were treated with PDB for 60 min to induce receptor down-regulation. The cells were subsequently washed free of PDB and resuspended in medium at 37°C. At different time points, aliquots of the cells were analyzed for receptor re-expression by flow cytometry. Comparable results were obtained in three independent experiments.

PP2A activation counteracts PKC-mediated TCR down-regulation

Given Equation 1 and the observations that PP2A and PKC activation increases k_out and k_in, respectively, it should be possible to define experimental settings in which simultaneous activation of PP2A and PKC leave TCR cell surface expression unaffected. These conditions give equations

(2)

(3)

where k_in' is the increased rate constant in following PKC activation and k_out' is the increased rate constant out following PP2A activation. We have previously shown that k_in can be finely regulated by adjustment of PKC activity and that new stable levels of TCR expression are obtained by incubation of T cells with different concentrations of PDB (9). To examine how PP2A activation affected TCR expression in cells with different k_in, Jurkat cells were preincubated with various concentrations of PDB for 1 h and subsequently incubated with either 25 µM C2 or medium for 30 min in the continuous presence of PDB. The cells were then washed and analyzed for TCR expression. Ceramide-induced activation of PP2A clearly counteracted PKC-mediated TCR down-regulation (Fig. 7). Interestingly, whereas isolated PP2A activation induced by 25 µM C2 mediated a 20% TCR up-regulation and isolated PKC activation induced by 10 nM PDB mediated a 30% TCR down-regulation, simultaneous PP2A and PKC activation using these concentrations of C2 and PDB left TCR expression unaffected (Fig. 7). This allowed us to calculate the distribution of [TCR]_s and [TCR]_i in the total pool of recycling TCR as follows.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. PP2A activation counteracts PKC-mediated TCR down-regulation. Cells were pretreated with various concentrations of PDB for 1 h and subsequently incubated with either 25 µM C2 or medium for 30 min in the continuous presence of PDB. The cells were then analyzed for TCR expression by flow cytometry. TCR expression was determined by comparing anti-CD3 MFI of PDB and PDB plus C2-treated cells with MFI of untreated cells. Comparable results were obtained in four independent experiments.

(4)

where z = [TCR]_i/[TCR]_s, a = ([TCR]_s - [TCR]_s')/[TCR]_s, and [TCR]_s' is the number of TCR expressed at the cell surface following down-regulation, given the assumption that k_out and the total number of TCR in the recycling pool are constant. Likewise, the relation between k_out' and k_out is described by equation

(5)

where b = ([TCR]_s'' - [TCR]_s)/[TCR]_s and [TCR]_s'' is the number of TCR expressed at the cell surface following up-regulation, given the assumption that k_in and the total number of TCR in the recycling pool are constant. During experimental conditions in which k_in and k_out are increased by the same factor and TCR expression accordingly is unaffected, Equations 3, 4, and 5 give

(6)

By insertion in Equation 6 of a = 0.3 (corresponding to 30% TCR down-regulation at 10 nM PDB) and b = 0.2 (corresponding to 20% TCR up-regulation at 25 µM C2), we get z = 0.375. This means that [TCR]_s total 1/(1 + 0.375) x 100% = 73% and [TCR]_i total 0.375/(1 + 0.375) x 100% = 27% of the entire pool of recycling TCR. These calculated values for the distribution of the TCR in the recycling pool agree well with previous experimental determinations (5, 6, 7, 28).

TCR up-regulation correlates with increased T cell responsiveness

It has been shown that a decrease in TCR expression levels leads to a reduction in the responsiveness of T cells following stimulation of the TCR (1, 2, 3). Consequently, we investigated whether PP2A-induced TCR up-regulation leads to an increase of T cell responsiveness following TCR ligation. We used induction of CD69 and CD25 expression as indicators for T cell activation. To determine whether a 15–20% TCR up-regulation affected T cell responsiveness, cells were pretreated with 25 µM C2 for 30 min to induce TCR up-regulation or were left untreated. The cells were subsequently washed three times to remove C2, resuspended in ice-cold medium containing anti-human TCR mAb, and incubated 30 min on ice. The cells were washed three times to remove unbound mAb and resuspended in 37°C medium. Following incubation for 22 h at 37°C, the cells were analyzed for CD69 and CD25 expression by flow cytometry. Pretreatment of the cells with C2 repeatedly resulted in a 10–15% increase in CD69 and CD25 surface expression following TCR stimulation as compared with nonpretreated cells (Fig. 8). Pretreatment with C2 without subsequent TCR stimulation did not induce CD69 or CD25 expression (data not shown). Thus, TCR up-regulation correlated with augmented T cell responsiveness following TCR ligation as measured by CD69 and CD25 expression.

View larger version (19K): [in this window] [in a new window]   FIGURE 8. TCR up-regulation correlates with increased T cell responsiveness. Cells were pretreated with 25 µM C2 for 30 min at 37°C to induce TCR up-regulation or were left untreated. The cells were subsequently washed free of C2 and incubated on ice with anti-human TCR mAb for 30 min. The cells were washed three times to remove unbound mAb and resuspended in 37°C medium. Following incubation for 22 h at 37°C, the cells were analyzed for (A) CD69 or (B) CD25 expression by flow cytometry. CD69/CD25 expression induced by TCR ligation of cells not pretreated with C2 was set to 100%. Results are given as mean ± SD of triplicate experiments. Comparable results were obtained in three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In general, TCR up-regulation can be mediated either by an increase of k_out, a decrease of k_in, or a combination of the two. As ceramide treatment did not affect k_in, ceramide-induced TCR up-regulation most probably was caused by an increase of k_out. This observation was further supported by the observation that ceramide counteracted the effect of PKC, which increases k_in. Based on these observations, the theoretical distribution of the TCR in the recycling pool could be calculated. We found that 75% of the recycling TCR were found at the cell surface and 25% were found inside the cell. These values are in good agreement with previous experimental determinations of TCR distribution (5, 6, 7, 28). From the present equations, it could also be calculated that the maximal possible TCR up-regulation was 33% (25/75 x 100% = 33%). As we repeatedly found that ceramide could induce 20–25% TCR up-regulation, we could conclude that the majority of intracellular TCR in the recycling pool was recruited to the plasma membrane following ceramide treatment.

The kinetics of ceramide-induced TCR up-regulation were comparable with the kinetics of ceramide-induced activation of phosphatases (29, 30), and experiments using protein phosphatase inhibitors demonstrated that the ceramide-induced TCR up-regulation most probably was mediated via ceramide-activated protein phosphatases belonging to the PP2A group. This is in agreement with previous studies demonstrating that ceramide is a potent activator of PP2A (17, 18). Furthermore, these results also concur with the observation that inhibition of PP2A leads to down-regulation of TCR expression (9). It has been reported that ceramide inactivates cellular PKC, and it could be speculated that the observed TCR up-regulation could be caused by an inhibition of PKC activity (31). However, inhibition of PKC was first observed following 4.5-h incubation with ceramide and could thus not explain the rapid TCR up-regulation observed in the present study. In addition, TCR up-regulation mediated by an inhibition of PKC should result in a decrease of k_in, which was not the case in the present study.

At least two distinct, independent pathways exist for down-regulation of the TCR. Ligand-induced TCR down-regulation is dependent on protein tyrosine kinases but independent of PKC and the CD3 L-based motif. In contrast, PKC-induced TCR down-regulation is dependent on the CD3 L-based motif but independent of protein tyrosine kinases (9). As opposed to ligand-induced TCR down-regulation that leads to TCR degradation, TCR down-regulated following PKC activation is not degraded but retained in intracellular vesicles until recycled back to the cell surface (3). Like PKC-mediated TCR down-regulation, ceramide-mediated TCR up-regulation was dependent on the CD3 L-based motif. This strongly suggested that the ceramide-PP2A pathway operates on the recycling pool of TCR. Thus, ceramide-induced activation of PP2A influences the balance between TCR expressed at the cell surface and TCR retained in intracellular vesicles in favor of the first. Our results support the hypothesis that TCR expression levels are controlled and can be finely tuned by the balance between PKC and PP2A activities (Fig. 9). Whereas the molecular mechanisms and the CD3 L-based motif involved in TCR internalization are well-characterized (2, 8, 28), the mechanisms and motifs involved in TCR exocytosis still remain to be determined. The present study of chimeric CD16-CD3 molecules associated with the -chain demonstrated that neither the CD3 L-based motif nor motifs in the -chain were sufficient to mediate exocytosis.

View larger version (12K): [in this window] [in a new window]   FIGURE 9. Model for TCR recycling. A, In unstimulated T cells a basic level of TCR recycling takes place. The distribution of the TCR in the recycling pool is in equilibrium with 75% TCR at the plasma membrane and 25% TCR in the intracellular pool. B, During circumstances with increased PKC activity the kin' is increased compared with the kin of untreated cells. A new TCR equilibrium is reached with a reduced number of TCR expressed at the plasma membrane and an increased number of TCR found in intracellular vesicles. C, Conversely, during circumstances with increases PP2A activity the kout' is increased compared with the kout of untreated cells. A new TCR equilibrium is reached with an increased number of TCR expressed at the plasma membrane and a reduced number of TCR found in intracellular vesicles.

	Acknowledgments

 
We thank Bodil Nielsen for technical assistance.

	Footnotes

 
¹ This work was supported by the Danish Cancer Society, the Danish Medical Research Council, the Carlsberg Foundation, Astrid Thaysens Foundation for Basic Medical Research, Director Leo Nielsens Foundation for Basic Medical Research, Lily Benthine Lunds’ Foundation, the Danish Medical Association Research Fund, and the Danish Biotechnological Research and Development Program. C.M. was recipient of a scholarship from Director Leo Nielsens Foundation for Basic Medical Research. J.P.H.L. was recipient of a scholarship from the Danish Cancer Society. J.K. was recipient of a Ph.D. scholarship from the University of Copenhagen. J.D. was recipient of a postdoctoral fellowship from the Danish Medical Research Council. A.-M.K.W. was recipient of a postdoctoral fellowship from the Carlsberg Foundation.

² Address correspondence and reprint requests to Dr. Carsten Geisler, Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, Building 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark.

³ Abbreviations used in this paper: PKC, protein kinase C; L-based motif, leucine-based receptor-sorting motif; PP2A, serine/threonine protein phosphatase 2A; C2, C₂-ceramide; DHC2, dihydro-C₂-ceramide; MFI, mean fluorescence intensity; PDB, phorbol 12,13-dibutyrate; WT, wild type.

Received for publication April 25, 2000. Accepted for publication July 3, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Viola, A., A. Lanzavecchia. 1996. T cell activation determined by T cell receptor number and tunable thresholds. Science 273:104.[Abstract]
2. Dietrich, J., J. Kastrup, B. L. Nielsen, N. Odum, C. Geisler. 1997. Regulation and function of the CD3 DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro. J. Cell Biol. 138:271.[Abstract/Free Full Text]
3. Dietrich, J., T. Backstrom, J. P. H. Lauritsen, J. Kastrup, M. D. Christensen, F. von Bulow, E. Palmer, C. Geisler. 1998. The phosphorylation state of CD3 influences T cell responsiveness and controls T cell receptor cycling. J. Biol. Chem. 273:24232.[Abstract/Free Full Text]
4. Dave, V. P., D. Allman, D. L. Wiest, D. J. Kappes. 1999. Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection. J. Immunol. 162:5764.[Abstract/Free Full Text]
5. Minami, Y., L. E. Samelson, R. D. Klausner. 1987. Internalization and cycling of the T cell antigen receptor. J. Biol. Chem. 262:13342.[Abstract/Free Full Text]
6. Krangel, M. S.. 1987. Endocytosis and recycling of the T3-T cell receptor complex: the role of T3 phosphorylation. J. Exp. Med. 165:1141.[Abstract/Free Full Text]
7. Boyer, C., N. Auphan, F. Luton, J.-M. Malburet, M. Barad, J.-P. Bizozzero, H. Reggio, A.-M. Schmitt-Verhulst. 1991. T cell receptor/CD3 complex internalization following activation of a cytolytic T cell clone: evidence for a protein kinase C-independent staurosporine-sensitive step. Eur. J. Immunol. 21:1623.[Medline]
8. Dietrich, J., X. Hou, A.-M. K. Wegener, C. Geisler. 1994. CD3 contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor. EMBO J. 13:2156.[Medline]
9. Lauritsen, J. P. H., M. D. Christensen, J. Dietrich, J. Kastrup, N. Ø dum, C. Geisler. 1998. Two distinct pathways exist for down-regulation of the TCR. J. Immunol. 161:260.[Abstract/Free Full Text]
10. Tonnetti, L., M. C. Veri, E. Bonvini, L. D’Adamio. 1999. A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction. J. Exp. Med. 189:1581.[Abstract/Free Full Text]
11. Simarro, M., J. Calvo, J. M. Vila, L. Places, O. Padilla, J. Alberola-Ila, J. Vives, F. Lozano. 1999. Signaling through CD5 involves acidic sphingomyelinase, protein kinase C-, mitogen-activated protein kinase kinase, and c-Jun NH₂-terminal kinase. J. Immunol. 162:5149.[Abstract/Free Full Text]
12. Boucher, L. M., K. Wiegmann, A. Futterer, K. Pfeffer, T. Machleidt, T. W. Mak Schutze, M. Kronke. 1995. CD28 signals through acidic sphingomyelinase. J. Exp. Med. 181:2059.[Abstract/Free Full Text]
13. Mathias, S., A. Younes, C. C. Kan, I. Orlow, C. Joseph, R. N. Kolesnick. 1993. Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cell-free system by IL-1ß. Science 259:519.[Abstract/Free Full Text]
14. Dbaibo, G. S., L. M. Obeid, Y. A. Hannun. 1993. Tumor necrosis factor-alpha (TNF-) signal transduction through ceramide: dissociation of growth inhibitory effects of TNF- from activation of nuclear factor- B. J. Biol. Chem. 268:17762.[Abstract/Free Full Text]
15. Schutze, S., K. Potthoff, T. Machleidt, D. Berkovic, K. Wiegmann, M. Kronke. 1992. TNF activates NF-B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71:765.[Medline]
16. Hannun, Y. A.. 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269:3125.[Free Full Text]
17. Dobrowsky, R. T., C. Kamibayashi, M. C. Mumby, Y. A. Hannun. 1993. Ceramide activates heterotrimeric protein phosphatase 2A. J. Biol. Chem. 268:15523.[Abstract/Free Full Text]
18. Galadari, S., K. Kishikawa, C. Kamibayashi, M. C. Mumby, Y. A. Hannun. 1998. Purification and characterization of ceramide-activated protein phosphatases. Biochemistry 37:11232.[Medline]
19. Schissel, S. L., E. H. Schuchman, K. J. Williams, I. Tabas. 1996. Zn²⁺-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J. Biol. Chem. 271:18431.[Abstract/Free Full Text]
20. Ballou, L. R., S. J. Laulederkind, E. F. Rosloniec, R. Raghow. 1996. Ceramide signalling and the immune response. Biochim. Biophys. Acta 1301:273.[Medline]
21. Perry, D. K., Y. A. Hannun. 1998. The role of ceramide in cell signaling. Biochim. Biophys. Acta 1436:233.[Medline]
22. Koretzky, G. A., J. Picus, T. Schultz, A. Weiss. 1991. Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc. Natl. Acad. Sci. USA 88:2037.[Abstract/Free Full Text]
23. Geisler, C.. 1992. Failure to synthesize the CD3- chain: consequences for T cell antigen receptor assembly, processing, and expression. J. Immunol. 148:2437.[Abstract]
24. Dietrich, J., C. Geisler. 1998. T cell receptor allows stable expression of receptors containing the CD3 leucine-based receptor-sorting motif. J. Biol. Chem. 273:26281.[Abstract/Free Full Text]
25. Geisler, C., T. Plesner, G. Pallesen, K. Skjodt, N. Odum, J. K. Larsen. 1988. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01. Scand. J. Immunol. 27:685.[Medline]
26. Geeraert, L., G. P. Mannaerts, P. P. van Veldhoven. 1997. Conversion of dihydroceramide into ceramide: involvement of a desaturase. Biochem. J. 327:125.
27. Fabbri, M., L. Fumagalli, G. Bossi, E. Bianchi, J. R. Bender, R. Pardi. 1999. A tyrosine-based sorting signal in the ß₂ integrin cytoplasmic domain mediates its recycling to the plasma membrane and is required for ligand-supported migration. EMBO J. 18:4915.[Medline]
28. Dietrich, J., X. Hou, A.-M. K. Wegener, L. O. Pedersen, N. Odum, C. Geisler. 1996. Molecular characterization of the di-leucine based internalization motif of the T cell receptor. J. Biol. Chem. 271:11441.[Abstract/Free Full Text]
29. Wolff, R. A., R. T. Dobrowsky, A. Bielawska, L. M. Obeid, Y. A. Hannun. 1994. Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction. J. Biol. Chem. 269:19605.[Abstract/Free Full Text]
30. Reyes, J. G., I. G. Robayna, P. S. Delgado, I. H. Gonzalez, J. Q. Aguiar, F. E. Rosas, L. F. Fanjul, C. R. Galarreta. 1996. c-Jun is a downstream target for ceramide-activated protein phosphatase in A431 cells. J. Biol. Chem. 271:21375.[Abstract/Free Full Text]
31. Lee, J. Y., Y. A. Hannun, L. M. Obeid. 1996. Ceramide inactivates cellular protein kinase C. J. Biol. Chem. 271:13169.[Abstract/Free Full Text]
32. Hofmeister, R., K. Wiegmann, C. Korherr, K. Bernardo, M. Kronke, W. Falk. 1997. Activation of acid sphingomyelinase by interleukin-1 (IL-1) requires the IL-1 receptor accessory protein. J. Biol. Chem. 272:27730.[Abstract/Free Full Text]
33. Kolesnick, R., D. W. Golde. 1994. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 77:325.[Medline]
34. Hannun, Y. A.. 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. Biol. Chem. 269:3125.
35. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395:82.[Medline]
36. Valitutti, S., S. Muller, M. Cella, E. Padovan, A. Lanzavecchia. 1995. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375:148.[Medline]
37. Valitutti, S., A. Lanzavecchia. 1997. Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition. Immunol. Today 18:299.[Medline]

This article has been cited by other articles:

				U. D'Oro, I. Munitic, G. Chacko, T. Karpova, J. McNally, and J. D. Ashwell Regulation of Constitutive TCR Internalization by the {zeta}-Chain J. Immunol., December 1, 2002; 169(11): 6269 - 6278. [Abstract] [Full Text] [PDF]

				M. von Essen, C. Menne, B. L. Nielsen, J. P. H. Lauritsen, J. Dietrich, P. S. Andersen, K. Karjalainen, N. Odum, and C. Geisler The CD3{gamma} Leucine-Based Receptor-Sorting Motif Is Required for Efficient Ligand-Mediated TCR Down-Regulation J. Immunol., May 1, 2002; 168(9): 4519 - 4523. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Menné, C. Articles by Geisler, C. Search for Related Content PubMed PubMed Citation Articles by Menné, C. Articles by Geisler, C.