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Secretion of IL-2 and IFN-, But Not IL-4, by Antigen-Specific T Cells Requires Extracellular ATP¹

Heather P. Langston^*, Yong Ke^2,*, Andrew T. Gewirtz, Kenneth E. Dombrowski^, and Judith A. Kapp^3,*,

Departments of ^* Ophthalmology, Biochemistry and Microbiology/Immunology, and Pathology, Emory University School of Medicine, Atlanta, GA 30322; and Atlanta Research and Education Foundation and Department of Veterans Affairs Medical Center, Decatur, GA 30033

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Extracellular ATP and other nucleotides transmit signals to cells via surface-associated molecules whose binding sites face the extracellular milieu. Ecto-nucleoside triphosphate diphosphohydrolase is such an ATP-binding enzyme that is expressed by activated lymphocytes. We have previously shown that nonhydrolyzable ATP analogs block the lytic activity of NK cells and CD8⁺ T cells as well as their E-NTPDase activity. These results suggest that the hydrolysis of ATP may play a role in lymphocyte function. Here we report that E-NTPDase activity is up-regulated within 15 min of T cell stimulation and that reversible and irreversible enzyme inhibitors profoundly reduce secretion of IL-2 and IFN-, but not IL-4. TNF-, IL-10, and IL-5 production showed intermediate sensitivity to these ATP analogs. Depletion of extracellular ATP also inhibited secretion of IFN-, but not IL-4, supporting the interpretation that extracellular ATP is required for secretion of some, but not all, cytokines. E-NTPDase antagonists reduced transcription of IL-2 mRNA and inhibited TCR-mediated intracellular calcium flux. These results suggest that extracellular ATP plays an essential role in the TCR-mediated signal transduction cascade for expression of certain cytokine genes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adenosine triphosphate is a normal component of the extracellular milieu that exerts a variety of physiological effects on hematopoietic and nonhematopoietic cells. Extracellular ATP does not cross the cell membrane, but rather mediates its biological actions through interaction with specific transmembrane proteins on the cell surface (1, 2, 3). Several proteins on the cell surface have been described that are capable of binding extracellular ATP and other nucleotides. Purinergic (P2)⁴ receptors bind ATP and other nucleotides transmitting signals to the cells, because these receptors have either intrinsic ion channel activity (P2X) or are coupled to G-proteins (P2Y) (4). Other ATP-binding transmembrane proteins are hydrolytic enzymes that are referred to as ecto-enzymes.

Until recently, it was thought that individual ecto-enzymes expressed specificity for individual tri- or diphosphorylated nucleotides; consequently, they were referred to as ecto-adenosine triphosphatase (ecto-ATPase), ecto-adenosine diphosphatase (ecto-ADPase), and so forth. The cloning of several genes encoding these enzymes revealed that they have overlapping substrate specificities (reviewed in Ref. 5). Thus, several families of ecto-nucleotidases are now recognized and described by a newly proposed nomenclature (6), which will be used in this study. The ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family hydrolyzes both purine and pyrimidine nucleoside tri- and diphosphates. The ectonucleotide pyrophosphatase/phosphodiesterase family has a broad substrate specificity and possesses phosphodiesterase as well as nucleotide pyrophosphatase activity. Ecto-alkaline phosphatases release inorganic phosphate (P_i) from a variety of organic compounds, including nucleotides. Ecto-5'-nucleotidase (CD73) hydrolyzes nucleoside 5'-monophosphates to the nucleoside derivative. The E-NTPDase family can be distinguished from the other ecto-enzymes that hydrolyze ATP by the reaction products they release: E-NTPDase releases AMP and P_i, ectonucleotide pyrophosphatase/phosphodiesterase releases AMP and pyrophosphate, and ecto-alkaline phosphatase releases adenosine and P_i. E-NTPDase is also distinct from ecto-5'-nucleotidase (CD73), which releases adenosine and P_i from AMP, but does not hydrolyze ATP.

E-NTPDases are distinct from all other families of ion-transport ATPases, which have their active sites located in the cytoplasm of the cell and hydrolyze only ATP (7). E-NTPDases, as defined by ecto-ATPase activity, are expressed by a variety of tissue types, including lymphocytes (7); however, the physiological role of this enzyme in lymphocytes is not well understood. By measuring the release of -³²P_i from [-³²P]ATP, activated human B cells (8), EBV-transformed human B cells (9), human NK cell lines (10), and activated CD8⁺ CTLs from mice (11) hydrolyze extracellular ATP. Greater than 90% of the ATPase activity is cell associated and thus is not due to enzymes secreted by or released from the cytoplasm (9, 11, 12). In addition, the hydrolysis of ATP by activated CD8⁺ T cells is resistant to fluoride, a phosphatase inhibitor, and these cells have no detectable ecto-5'-nucleotidase activity (11). This enzymatic activity is classified as an E-NTPDase as defined by three distinct criteria (7): 1) Ca²⁺ or Mg²⁺ dependence (12); 2) insensitivity to inhibitors for P-type, F-type, and V-type ATPases (9, 11, 12); and 3) the ability to hydrolyze several nucleoside tri- and/or diphosphates (9, 12).

We have also demonstrated that nonhydrolyzable ATP analogs block the cytolytic activity of the human NK3.3 cell line for NK-sensitive targets (10) and Ab-sensitized targets (13). However, these analogs did not prevent conjugate formation between the NK3.3 and target cells (10), suggesting that ATP is not required for effector-target cell binding, but acts at a subsequent stage of activation. The cytolytic activity and secretion of IFN- and TNF- by CD8⁺ T cells was also blocked by nonhydrolyzable ATP analogs (11). Inhibition of CD8⁺ T cell functions by ATP analogs cannot be attributed to missing hydrolytic products because inhibition was not reversed by the addition of ADP or AMP (11). These observations suggest that extracellular ATP is required for certain lymphocyte functions.

In this study, the role of extracellular ATP in the regulation of additional effector T cell functions was investigated. We determined that E-NTPDase is rapidly up-regulated in T cells and that secretion of IL-2 and IFN-, but not IL-4, is dependent on extracellular ATP. We also demonstrated that a nonhydrolyzable analog of ATP, adenosine 5'-[-thio]triphosphatate (ATPS), inhibited cytokine expression at the level of mRNA. Moreover, ATPS inhibited TCR-mediated intracellular calcium flux, suggesting that extracellular ATP plays a role in calcium flux that is induced by interaction of TCR and Ag.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals and reagents

Female BALB/c and C57BL/6 (B6) mice were obtained from the National Cancer Institute (Frederick Cancer Facility, Frederick, MD). The TCR-1 TCR transgenic mice express the V2, V5 TCR derived from the K^b-restricted OVA_257–264-specific CTL clone 149.42 (14). Purified chicken egg OVA (grade VI), bovine insulin, ATPS, ,-methylene adenosine 5'-diphosphate, and apyrase (EC 3.6.1.5; grade VI) were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human insulin was generously provided by Eli Lilly (Indianapolis, IN). OVA_323–339 peptide was synthesized by the Microchemical Facility of Emory University (Atlanta, GA). N,N'-dimethylformamide (DMF) was purchased from Aldrich (Milwaukee, WI). 5'-p-(Fluorosulfonyl)benzoyl adenosine (5'-FSBA) was synthesized by condensation of adenosine with p-fluorosulfonylbenzoyl chloride as described by Pal et al. (15). PMA and ionomycin were purchased from Calbiochem (San Diego, CA).

Cell lines

OT4H.1D5 and OT4H.2D5 are CD4⁺ T cell hybridomas that were produced by fusion of BW5147^-- (16) with spleen cells from B6 mice primed with OVA in CFA (17). Both recognize OVA_265–280 in the context of I-A^b. OT8H.4B10 is a CD8⁺ T cell hybridoma that recognizes K^b-restricted OVA_257–264 (17). The CD4⁺ T cell clones BB68.3, BB80/12.7, and BB100.C4.30 were produced from spleen cells of B6 mice primed with bovine insulin in CFA (18). The CD4⁺ clone DH76/2.5 was derived from BALB/c mice primed with human insulin in CFA (18). CD4⁺ Th0, Th1, and Th2 clones specific for the OVA_323–339 epitope were generated from transgenic BALB/c mice expressing the DO11.10 TCR (19, 20) (provided by R. P. Bucy, University of Alabama, Birmingham, AL). OVA-specific (21) and bovine insulin-specific (17) CTL clones were produced by immunizing mice with 100 µg of Ag in CFA as previously described. Syngeneic spleen cells or the B cell hybridoma cell line F8.11 was used as a source of APCs in cultures of CD4⁺ T cells. F8.11 was produced by fusing the hypoxanthine/aminopterin/thymidine-sensitive secretory IgG⁺ tumor line M12.4.1 (H-2^d) (22) with LPS-activated B6 spleen cells (23). F8.11 expresses MHC class I and class II proteins of the H-2^d and H-2^b haplotypes (23). EL4 transfected with the OVA gene (E.G7-OVA) (24) was used to activate OVA-specific CTLs, and EL4 transfected with the insulin gene (17) was used for insulin-specific CTLs. All cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS, 1 mM L-glutamine, 1 mM sodium pyruvate, 50 µM 2-ME, plus antibiotics at 37°C in 6% CO₂ in air and were mycoplasma free.

Ecto-ATPase assay

Ecto-ATPase activity was measured by the release of ³²P_i from [-³²P]ATP (Amersham, Arlington Heights, IL), as described previously (12). Briefly, 1 x 10⁴ T cells that were >90% viable by trypan blue dye exclusion were incubated in triplicate with 0.3 mM cold ATP and [-³²P]ATP used as a trace label in a final volume of 200 µl. After 20 min at 37°C, the amount of ³²P_i released into the supernatant fluid was determined after precipitation of the nucleotides with activated charcoal.

Treatment of cells with ATP

T cell clones and hybridomas were suspended in serum-free RPMI 1640 medium at 37°C and were pretreated with the irreversible inhibitor 5'-FSBA dissolved in DMF or with DMF alone, as described previously (11). Briefly, various concentrations of 5'-FSBA were tested in a final concentration of DMF that was held constant at 2.5%. The cells were incubated with DMF or 5'-FSBA at 37°C for 10 min to 1 h, as indicated, and were washed two to three times in RPMI 1640 containing 0.5% DMF to remove unbound 5'-FSBA. The pretreatment was done to avoid any inhibitory effects on the APCs. In other experiments, cells were cultured for 24 h or were pretreated for 1 h in the presence of various concentrations of the reversible ecto-ATPase antagonist ATPS. The viability of cells after the above treatments was routinely >90% by trypan blue dye exclusion.

Production and assay of cytokines

CD4⁺ T cell hybridomas and clones were activated by incubation with irradiated (3,000 rad) syngeneic spleen cells or irradiated (10,000 rad) F8.11 B cells and the appropriate Ag. CD8⁺ T cell clones were activated with irradiated (20,000 rad) E.G7-OVA or EL4 transfected with the insulin gene as stimulators. After overnight incubation, supernatant fluids were collected and assayed for cytokine content by ELISA. In some experiments, the T cell hybridomas were incubated for 24 h in the presence of 1 µg/ml plate-bound anti-CD3 (145-2C11; BD PharMingen, San Diego, CA). Cytokines were quantitated by ELISA using mAb pairs (25). Microtiter plates were coated with capture mAb (BD PharMingen) overnight at 4°C and were blocked with PBS plus 1% BSA at 22°C for 1 h. Serially diluted, recombinant cytokines and test supernatant fluids were added in duplicate and incubated overnight at 4°C. Biotinylated detecting mAb and avidin-HRP conjugate (Vector Laboratories, Burlingame, CA) were added and incubated at 22°C for 45 min and 30 min, respectively. Plates were washed extensively between steps. The colorimetric reaction was developed by adding the substrate 2,2'-azino-di(3-ethyl-benzthiazoline sulfonate) (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Absorbance was read at 405 nm. Cytokine concentrations in supernatant fluids were calculated from the standard curve for the appropriate recombinant cytokine.

Fluorescent staining

Cells were treated with medium alone, DMF, or 5'-FSBA in DMF for 1 h, as described above. After washing, normal rabbit serum or rabbit anti-sulfonylbenzoyladenosine (anti-SBA) Ab (a gift from A. R. Beaudoin, University of Sherbrooke, Quebec, Canada) (26, 27, 28) was added to cells and incubated at 4°C for 30 min. Anti-SBA is directed against the protein-bound SBA moiety that forms after the electrophilic fluorosulfonyl group of 5'-FSBA reacts with protein. The SBA anti-serum was generated by immunization with one FSBA-modified nucleotide-binding protein and boosting with different FSBA-modified nucleotide-binding carrier proteins (28). The cells were washed, and biotinylated goat anti-rabbit IgG (Vector Laboratories) was added as secondary Ab followed by PE-streptavidin (Biomedia, Foster City, CA). FACS analysis was performed with a FACScan flow cytometer (BD Biosciences, San Jose, CA). Dead and aggregated cells were excluded by forward and side light scatter. Results are presented as fluorescence histograms with cell number on the y-axis and log fluorescence intensity on the x-axis.

RNase protection assay

RNAzol B (Tel-Test, Friendswood, TX) was used to isolate total RNA from 5 x 10⁶ T cell hybridomas that had been stimulated with plate-bound anti-CD3 in the presence of 0.1 mM ATPS or 0.5 mM ATPS or left untreated. The Riboquant multiprobe RNase protection assay system (BD PharMingen) was used to analyze the expression of the cytokines IL-2 and IL-4. A ³²P-labeled anti-sense RNA from each cytokine was hybridized to the target RNA. After treatment with RNase to remove any single-stranded RNA, the probe and target RNA were resolved by denaturing PAGE. [-³²P]UTP (3000 Ci/mmol, 10 mCi/ml) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Results were visualized by autoradiography and the bands were quantitated using a phosphor imager (Amersham Pharmacia Biotech).

Intracellular calcium measurements

DO11.10 Th1 cells were incubated at 37°C with 2 µM fura 2-AM (Molecular Probes, Eugene, OR) and 30 µg/ml anti-CD3 for 30 min in phenol red-deficient HBSS. After loading, cells were washed and maintained at room temperature for the duration of the experiment. Approximately 2.25 x 10⁵ fura 2-loaded Th1 cells were added to a cuvette containing HBSS. The cuvette was then placed in a spectrofluorometer (model F-4500; Hitachi, Tokyo, Japan) equipped with a stirring system and a thermostat that was set to 37°C. Fluorescence was measured at 505 nm, while excitation wavelength was changed between 340 and 380 nm via Hitachi Intracellular Cation software. Baseline Ca²⁺ was measured for 1 min. Ca²⁺ levels of the cells in the control group were measured for an additional 5 min. In the experimental group, ATPS (final concentration, 0.5 mM) was added to the cuvette and Ca²⁺ levels of the cells were measured for 5 min. Next, the cells were activated by cross-linking the anti-CD3 by adding anti-hamster IgG (1:66.7; Vector Laboratories) to the cuvette. The changes in fluorescence due to stimulus addition were subtracted (29). Intracellular Ca²⁺ was calculated using the Grynkiewicz equation: (R - R_min)/(R_max - R) x K_d. R_max was measured as the fluorescence of cells after addition of 10 µg of ionomycin, and R_min was measured as the fluorescence of cells after addition of 50 mM EDTA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of ecto-ATPase activity by T cells

The ecto-ATPase activity of various T cell lines, clones, and hybridomas was measured directly by quantitating the release of -³²P_i from [-³²P]ATP. Naive splenic T cells had levels of activity indistinguishable from background (Table I). In contrast, the fusion partner for the hybridomas (BW5147) had very low but detectable levels of ecto-ATPase activity that were significantly above the negative controls. The CD4⁺ T cell hybridomas and the CD4⁺ T cell clones expressed significantly higher levels of enzymatic activity, which were comparable to those expressed by CD8⁺ T cells (11) but were somewhat lower than those of the human NK cell line NK3.3 (12). These data suggest that extracellular ATP could play a role in the function of CD4⁺ T lymphocytes.

Expression of ecto-ATPase activity by T cell clones and hybridomas, but not naive T cells, suggests that expression was activated by Ag. To determine when ecto-ATPase activity is induced upon antigenic stimulation, freshly isolated spleen cells from TCR-1 mice expressing a transgenic TCR specific for OVA were stimulated with E.G7-OVA cells, and the hydrolysis of extracellular ATP was measured at various time points. Splenic T cells from the TCR-1 transgenic mice were used in this experiment because 10% of the T cells express the V2, V5 TCR derived from the K^b-restricted, OVA_257–264-specific CTL clone 149.2 (14). The TCR transgenic T cells allow the detection of Ag-specific T cell responses, which is not possible in nontransgenic mice because of low precursor frequency of naive Ag-specific cells. Within 15 min after stimulation with irradiated E.G7-OVA cells, the naive CD8⁺ TCR-1 T cells expressed detectable ecto-ATPase activity (Fig. 1). Maximal activity was detected 30 min poststimulation, and it returned to background levels by 60 min. A similar pattern of enzymatic activation was observed upon restimulation of the transgenic T cells that had been in culture for 1 wk (Fig. 1). These data demonstrate that ecto-ATPase activity is rapidly induced on naive and previously activated T cells by Ag, suggesting that it could be involved in the regulation of T cell activation or effector functions.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Up-regulation of ecto-ATPase activity. Splenic T cells from naive TCR-1 transgenic mice () and TCR-1 transgenic splenic T cells that had been cultured for 1 wk () were incubated with irradiated (20,000 rad) E.G7-OVA cells. Ecto-ATPase activity was measured at the indicated time points as described in Materials and Methods. The background level of response in tubes with irradiated E.G7-OVA cells (2.87 pmol 32Pi) was subtracted from the values of the test cells for both experiments. The background level of response in tubes containing irradiated B6 spleen cells required for the second Ag stimulation was below the level indicated for the E.G7-OVA cells. Each sample was assayed in triplicate with results from a representative experiment shown.

To investigate whether effector functions of MHC class II-restricted, CD4⁺ T cells were dependent on extracellular ATP, they were treated with 5'-FSBA, which is an ATP analog that irreversibly inhibits E-NTPDase. Because 5'-FSBA binds at or near the active site of the enzyme (12), an anti-SBA Ab was used to visualize cell surface binding of 5'-FSBA by CD4⁺ T cells (26, 28). BW5147 showed a low level of staining with this Ab (Fig. 2A) with (solid line) or without (dotted line) 5'-FSBA, which is consistent with the relatively low amount of enzymatic activity shown in Table I. The T cell hybridomas OT4H.1D5 and OT4H.2D5 reacted with the Ab after treatment with 5'-FSBA in DMF (solid line), but not when treated with DMF alone (dotted line; Fig. 2A). OVA-specific, CD4⁺ Th0, Th1, and Th2 cell clones derived from transgenic mice expressing the DO11.10 TCR also reacted with the rabbit Ab after treatment with 5'-FSBA (solid line), whereas normal rabbit serum (dotted line) did not stain the T cell clones treated with 5'-FSBA (Fig. 2B). The results of the cell surface staining show a concordance of 5'-FSBA binding with the level of expression of ecto-ATPase activity as reported in Table I.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Binding of 5'FSBA by T cells. BW5147, OT4H.1D5, or OT4H.2D5 cells (A) were treated with 2.5% DMF (dotted line) or 0.5 mM 5'-FSBA (solid line) at 37°C for 60 min, then were stained with rabbit anti-SBA Ab followed by biotinylated goat anti-rabbit IgG and PE-streptavidin conjugate. Stained cells were analyzed by a FACScan flow cytometer and are shown as fluorescence histograms with the cell number on the y-axis and log fluorescence intensity on the x-axis. CD4+ Th0, Th1, and Th2 clones (B) from transgenic BALB/c mice expressing the DO11.10 TCR were treated with 0.25 mM 5'-FSBA in 2.5% DMF at 37°C for 60 min. The cells were washed and treated with normal rabbit serum (dotted lines) or rabbit anti-SBA Ab (solid lines). Stained cells were analyzed by a FACScan flow cytometer and are shown as fluorescence histograms with the cell number on the y-axis and log fluorescence intensity on the x-axis.

Extracellular ATP is required for the secretion of IFN- and IL-2, but not IL-4

5'-FSBA binds to CD4⁺ T cell lines as well as hybridomas, and it is an antagonist for E-NTPDase; hence, it was used to determine whether blocking the enzyme inhibited cytokine secretion by CD4⁺ T cells. DMF-treated control cells produced amounts of cytokines (Table II) comparable to those of untreated control cells (data not shown), indicating that the solvent was not inhibitory. 5'-FSBA treatment dramatically inhibited secretion of IFN- by the CD8⁺ OVA-CTLs (as previously shown in Ref. 11) and the CD4⁺ Th1 clone DH76/12.1. In contrast, 5'-FSBA did not inhibit IL-4 production by the CD4⁺ Th2 clones (DH 76/2.5 and BB68.3) and often enhanced IL-4 production (BB68.3). TNF- secretion was partially inhibited by 5'-FSBA in the three cell lines that expressed this cytokine. T cell clones capable of producing both type 1 and type 2 cytokines (BB100.C4.30, BB42.B23.5, BB80/12.7, and insulin-CTL) were particularly informative because IFN- production was profoundly inhibited by 5'-FSBA, whereas IL-4 production was not. Inhibition of IFN- but not IL-4 in the same T cell verifies that the irreversible inhibitor 5'-FSBA is not nonspecifically toxic in these short-term assays. These results suggest that E-NTPDase activity is required for the secretion of some, but not all, cytokines by nontransformed T cells.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Effect of 5'-FSBA on Th0, Th1, or Th2 T cell clones derived from DO11.10 TCR transgenic mice. T cell clones were treated with 2.5% DMF or 0.25 mM 5'-FSBA in DMF at 37°C for 60 min. After washing, 3 x 105 treated T cells were incubated with 3 x 106 syngeneic, irradiated (2000 rad) spleen cells as APCs with or without 1 µM OVA323–339 peptide at 37°C for 24 h. Supernatants were tested for IFN-, IL-4, IL-5, and IL-10 by cytokine ELISA. Results are shown as cytokine concentrations calculated from standard curves of the appropriate recombinant cytokine.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Effect of ATPS on T cell hybridomas. The T cell hybridomas OT4H.1D5 and OT4H.2D5 were treated with increasing concentrations of ATPS (A) or with or without 0.5 mM 5'-FSBA (B) and were cultured with 1 µg/ml plate-bound 2C11. After 24 h, supernatants were harvested and tested for IL-2 and IL-4 by cytokine ELISA. Results are shown as cytokine concentrations calculated from standard curves of the appropriate recombinant cytokine.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. ATPS is a reversible inhibitor of IFN- secretion. The Th1 T cell clone derived from DO11.10 TCR transgenic mice was incubated for 24 h on anti-CD3 (1 µg/ml) coated plates in the presence of the indicated concentrations of ATPS (•), or cells were pretreated with the indicated concentration of ATPS for 1 h and then were washed three times and incubated 24 h on anti-CD3 (1 µg/ml) coated plates (). Supernatants were tested for IFN- by ELISA. Results are shown as percent control responses in the absence of ATPS. Th1 cells produced CD3 and 56.3 ± 1.5 ng/ml IFN- after activation by plate-bound anti-CD3. Th1 cells treated with ATPS for 1 h produced 47.4 ± 0.9 ng/ml IFN- after activation by plate-bound anti-CD3.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Induction of IFN-, but not IL-4, requires extracellular ATP. The Th0 T cell clone derived from DO11.10 TCR transgenic mice was incubated with or without the indicated concentration of apyrase, syngeneic irradiated (2000 rad) spleen cells as APCs, and 1 µM OVA323–339 peptide at 37°C for 24 h. Supernatants were tested for IFN- and IL-4 by ELISA. Results are shown as percent control responses in the absence of apyrase. Control cultures of Th0 cells produced 3368 ± 186 pg/ml IFN- and 5470 ± 13 pg/ml IL-4 when stimulated with APCs and OVA323–339 peptide, but produced <50 pg/ml cytokines in the absence of Ag.

Secretion of cytokines requires protein synthesis (32), suggesting that T cells do not store cytokines. Thus, we hypothesize that extracellular ATP may be required for the activation of some cytokine genes but not others. To test this idea, the expression of mRNA levels of the relevant cytokine genes was determined by an RNase protection assay of T cells activated in the presence or absence of ATPS. The CD8⁺ T cell hybridoma OT8H.4B10 was cultured with or without ATPS during stimulation with 1 µg/ml plate-bound 2C11 (anti-CD3). After 4 h, the RNA was isolated and analyzed by RNase protection assay. ³²P-labeled probes from IL-2 and IL-4 were hybridized to the target RNA. After RNase treatment, the protected RNA was resolved by denaturing PAGE. Analysis of the RNA gel by autoradiography revealed a significant decrease in expression of IL-2 mRNA in the presence of 0.5 mM ATPS (Fig. 7A), whereas the expression of IL-4 mRNA increased slightly. Normalization of IL-2 and IL-4 mRNA to the housekeeping gene L32 supports this interpretation (Fig. 7B). Expression of IL-2 mRNA was decreased at the higher concentration of ATPS, whereas expression of IL-4 mRNA was increased. These data confirm that extracellular ATP is required for secretion of IL-2, but not IL-4, and suggest that this requirement is at the level of transcription.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Decreased expression of IL-2 mRNA, but not IL-4 mRNA, by ATPS. The CD8+ T cell hybridoma OT8H.4B10 was treated with 0.1 mM ATPS, 0.5 mM ATPS, or medium alone and was cultured with 1 µg/ml plate-bound 2C11. After 4 h, isolated RNA was analyzed by RNase protection assay (A). Signals were quantified by phosphor imager and the individual bands were normalized to the housekeeping gene L32 from the same sample (B).

The rapid induction of E-NTPDase after activation of naive T cells suggests that this enzyme might play a role in the TCR-mediated signal transduction cascade. We have previously reported that 5'-FSBA inhibits the influx of extracellular Ca²⁺ induced by cross-linking of the FcRIII on NK3.3 cells by anti-CD16 (10). 5'-FSBA also inhibits the spontaneous uptake of extracellular Ca²⁺ by EBV-transformed B cells (31), suggesting a potential role for E-NTPDase activity in calcium flux. To test this hypothesis, PMA and ionomycin were added to the T cell hybridoma OT4H.2D5 that was activated by plate-bound anti-CD3 in the continuous presence of ATPS. ATPS inhibited anti-CD3-induced IL-2 secretion by T cells, and the addition of 2.5 ng/ml PMA and 0.1 µM ionomycin overcame the inhibition (Fig. 8A). Concentrations of PMA and ionomycin that are routinely used to activate T cells (20 ng/ml and 1 µM, respectively) were toxic to the hybridoma when grown in the presence of ATPS (data not shown). At the lower concentrations used in Fig. 8, neither PMA nor ionomycin individually overcame the ATPS-mediated inhibition of IL-2 secretion, but the combination did so. There was no effect of ATPS or PMA and ionomycin on anti-CD3-induced IL-4 production (Fig. 8B). These data suggest that E-NTPDase may play a role in cytokine secretion through TCR-mediated calcium flux.

View larger version (14K): [in this window] [in a new window]   FIGURE 8. Effect of calcium ionophore on inhibition of cytokine secretion by ATPS. The T cell hybridoma OT4H.2D5 was cultured with 1 µg/ml plate-bound 2C11 with or without 0.5 mM ATPS, 0.1 µM ionomycin, and 2.5 ng of PMA, as indicated. After 16–24 h, supernatants were harvested and tested for IL-2 and IL-4 by cytokine ELISA. Results are shown as cytokine concentrations calculated from standard curves of the appropriate recombinant cytokine.

View larger version (27K): [in this window] [in a new window]   FIGURE 9. Inhibition of intracellular calcium flux by ATPS. CD4+ Th1 cells were coated with anti-CD3 and loaded with fura 2-AM. Cells were activated by cross-linking the anti-CD3 with anti-hamster IgG () without (A) or with 0.5 mM ATPS added () to the cells before cross-linking (B). Increases in intracellular calcium concentration were measured with a spectrofluorometer (at 37°C). To verify proper loading of the cells with fura 2-AM, ionomycin was added () followed by EDTA (). Fluorescence emission was read at 505 nm, while the excitation wavelength was changed between 340 and 380 nm. The concentration of intracellular Ca2+ was calculated using the Grynkiewicz equation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

5'-FSBA, an affinity label analog of ATP, was used to investigate the role of E-NTPDase in lymphocyte function. 5'-FSBA contains a reactive fluorosulfonyl group at a position that is similar to that of the or phosphoryl groups of ATP. Once 5'-FSBA is bound to the enzyme, electrophilic substitution of the fluoro group by nucleophilic amino acid side chains of the enzyme forms a covalent bond with the enzyme (33), thereby making it an irreversible antagonist of E-NTPDase activity. All of the activated T cells that were tested expressed E-NTPDase activity, yet 5'-FSBA inhibited secretion of IL-2 and IFN-, but not IL-4. IL-4 secretion was resistant to 5'-FSBA even in T cells in which IL-2 or IFN- secretion was simultaneously inhibited. Secretion of TNF-, IL-5, and IL-10 was intermediate in sensitivity to 5'-FSBA. T cells do not store cytokines, as judged by the observation that their secretion requires protein synthesis (32). Therefore, these results suggest that E-NTPDase activity may be required for Ag-dependent expression of some cytokine genes but not others. The possibility that differences in sensitivity of various T cells to 5'-FSBA might have resulted from heterogeneity of the TCR used by different clones was ruled out by showing that Th1, Th2, and Th0 subsets expressing identical DO11.10 TCR displayed the same patterns of sensitivity to 5'-FSBA as did nontransgenic T cell subsets. The inhibition of some but not all cytokines produced by CD4⁺ DO11.10 Th0 cells suggests that adhesion between T cells and Ag-bearing APCs is sufficient for some level of activation. Thus, the evidence presented in this manuscript favors the interpretation that E-NTPDase activity is required for secretion of some, but not all, cytokines by T cells.

The inhibition of E-NTPDase can be blocked by treating T cells with 5'-FSBA in the presence of excess of reversible antagonists, such as adenosine 5'-(,-imido)triphosphate and ATPS, which are competitive inhibitors of the enzyme with respect to ATP (12). Thus, 5'-FSBA binds to and inhibits E-NTPDase under the test conditions used. However, because 5'-FSBA is chemically reactive, it is conceivable that nonspecific binding of 5'-FSBA to some other cell surface protein might be responsible for the inhibition of cytokine secretion. We believe the latter possibility to be unlikely because other nonhydrolyzable ATP analogs that do not form covalent bonds, such as ATPS and adenosine 5'-(,-imido)triphosphate, also inhibit cytokine secretion. Our results with ATPS confirm and extend the results reported by Duhant et al. (34) showing that ATPS inhibits production of IL-2, IL-5, IL-10, and IFN- by human CD4⁺ T cells, which were activated with anti-CD3 and anti-CD28 Abs. Inhibition of type 1 cytokines by 5'-FSBA was more complete than was inhibition by ATPS. This is consistent with the fact that 5'-FSBA irreversibly modifies the ATP binding site, whereas ATPS is a reversible inhibitor that competes with ATP such that not all of the enzymatic activity is eliminated at doses that are not toxic to the cells. Depletion of the extracellular substrate, ATP, also inhibited IFN- but not IL-4 secretion. Thus, we conclude from these two independent approaches that extracellular ATP provides an essential signal for the activation of some, but not all, cytokines.

The source of the extracellular ATP in these cultures is probably the T cells. Accumulation of extracellular ATP was detected within 3 min of the activation of purified T cells with anti-CD3 (35). No ATP was detected in the cell supernatants, suggesting that the extracellular ATP was rapidly bound to surface proteins such as E-NTPDase or purinergic receptors. E-NTPDases hydrolyze ATP and ADP (6, 7). Therefore, nonhydrolyzable ATP analogs might inhibit a subsequent ecto-ADPase-dependent event by eliminating the substrate (ADP). However, an ADP analog, ,-methylene adenosine 5'-diphosphate, did not inhibit the secretion of either IL-2 or IL-4 by the OT4H.2D5 hybridoma (data not shown), suggesting that it is not the requirement for the reaction product that is essential for cytokine secretion. We previously demonstrated that 5'-FSBA inhibition of CTL activity could not be restored by the addition of AMP or adenosine (11). Furthermore, AMP cannot be formed from 5'-FSBA; therefore, AMP would not be activating P1 receptors under the conditions used in these experiments. Because nonhydrolyzable ATP analogs inhibit type 1 cytokine secretion, we favor the interpretation that the hydrolysis of extracellular ATP is essential for this pathway of activation. However, the possibility that ATP analogs might bind to E-NTPDase somewhat differently than ATP and thereby might transmit subtly different signals to the cell cannot be ruled out.

Although the activated, murine T cells that we have studied do not express other ecto-enzymes that hydrolyze ATP, P2 receptors that bind ATP and induce calcium flux have been detected on T cells (reviewed in Ref. 58). However, P2 receptors are not likely to be the direct targets for the ATP analogs for two reasons. First, 5'-FSBA inhibits E-NTPDase activity and type 1 cytokine secretion, but it does not agonize (or antagonize) P2 receptors (37). Second, ATPS inhibits E-NTPDase activity (36, 38) and secretion of type 1 cytokines, whereas ATPS is an agonist of P2 receptors (4, 39). Thus, inhibition of cytokine secretion correlates with the antagonism of E-NTPDase rather than the ability to activate P2 receptors. This interpretation also offers a potential explanation of the results reported by Duhant et al. (34), who found that cytokine production by human CD4⁺ T cells was inhibited by ATPS, which also induced an accumulation of cAMP by both naive and activated T cells. The authors thought that ATPS might be functioning as a P2 agonist, but it failed to induce calcium flux in CD4⁺ T cells. Furthermore, the CD4⁺ T cells did not express P2Y₁₁ receptors, which could account for the increase in cAMP because they are the only P2 receptors that are coupled to both adenylyl cyclase and phospholipase C pathways (38, 40). Our data suggest that E-NTPDase might be the target for ATPS-induced inhibition of cytokine secretion by activated CD4⁺ T cells in the study reported by Duhant et al. (34).

Recently, it has been reported that CD39, an early activation Ag expressed by B and T cells (41, 42), is a member of the E-NTPDase family (26, 43). The ecto-ADPase activity of CD39 is responsible for thromboregulation in humans (44, 45). CD39 is a 70- to 100-kDa glycoprotein that is also expressed by activated lymphoid cells (42, 46). However, the function of CD39 on lymphocytes has not yet been clearly defined. Thus, CD39 may be responsible for the hydrolysis of extracellular ATP, which appears to be required for secretion of type 1 cytokines by murine lymphocytes. Studies are underway to investigate this possibility.

The signaling pathways for cytokine secretion by T cells are very complex and not yet completely characterized. However, it is evident that cytokine genes are differentially regulated. For example, cytokine production by Th1 cells is more sensitive to inhibition by cholera toxin (47), cyclosporin A, and 8-bromo-cAMP than is cytokine production by Th2 cells (48, 49). PGE₂, which elevates intracellular cAMP, inhibits cytokine production by Th1 cells but not by Th2 cells (50). Moreover, IL-4 production by Th2 cell lines has been reported to be less dependent on Ca²⁺ flux than IL-2 production by Th1 cells (49, 51). We suggest that secretion of IL-4 by activated T cells may be relatively resistant to E-NTPDase inhibitors because the expression of the gene for this cytokine is less Ca²⁺ dependent than are the genes for IL-2, IFN-, and TNF-. Less information is available about regulation of IL-5 and IL-10 gene expression, but our results indicate that these genes are less sensitive to E-NTPDase inhibitors than are the genes for IL-2 or IFN-, but they are more sensitive than the IL-4 gene. Cytokine gene expression is regulated by a series of highly orchestrated proteins that bind to DNA elements to activate and/or inhibit transcription. Analysis of cytokine gene expression by RNase protection assay revealed the inhibition of IL-2 mRNA expression, but not IL-4 mRNA expression, in cells treated with ATPS. Hence, the differential sensitivity of cytokine gene expression to E-NTPDase antagonists may be useful in the identification of critical differences in the factors regulating induction of these cytokine genes.

Previous studies showed that 5'-FSBA inhibited the influx of extracellular ⁴⁵Ca²⁺ induced by cross-linking of the FcRIII on NK3.3 cells by anti-CD16 (10). Because receptor-mediated activation of T cells and B cells is also dependent upon sustained influx of extracellular Ca²⁺ (52, 53, 54), we asked whether ionomycin and PMA could overcome the ATPS-mediated inhibition of IL-2 secretion from the T cell hybridomas. Ionomycin induces capacitative calcium entry by depleting intracellular calcium stores (55, 56), whereas PMA induces release of calcium from intracellular stores by activating protein kinase C or p21^ras (57). The requirement for both PMA and ionomycin to reverse the inhibitory effects of ATPS suggests that ecto-ATPase might be regulating TCR-mediated intracellular signaling at two distinct steps. However, the concentrations of PMA and ionomycin, which reversed the inhibition by ATPS, were suboptimal for activating T cells. Thus, inhibition of any one pathway by ATPS could be sufficient to prevent reconstitution of the response under these conditions. We also showed that ATPS inhibited receptor-mediated intracellular calcium flux. Because ATPS-mediated inhibition of IL-2 secretion can be overcome by increasing Ca²⁺ with PMA and ionomycin, we speculate that the hydrolysis of extracellular ATP by E-NTPDase may be involved directly or indirectly in the regulation of Ca²⁺ flux that is induced by ligand binding to receptors on lymphocytes.

	Acknowledgments

 
We thank Ms. Linda M. Kapp, Mr. Michael Edgerton, and Mr. Sean Lyons for excellent technical assistance. We are very grateful to our colleagues who generously provided valuable cells and reagents, including Dr. R. Pat Bucy for providing DO11.10 TCR transgenic CD4⁺ T cell clones, Dr. Maurice Gately (Hoffmann-La Roche, Nutley, NJ) for providing recombinant human IL-2, Ms. Neidenthal (Eli Lilly) for providing recombinant human insulin, and Dr. Adrien Beaudoin for providing the rabbit anti-SBA serum.

	Footnotes

 
¹ This work was supported by Core Grant P30 EYO06360 from the National Eye Institute, National Institutes of Health; Research to Prevent Blindness; Department of Army Career Development Award DAMD17-94-J-4161 (to K.E.D.); Department of Army Grant DAMD17-94-J-4272 (to K.E.D.); Department of Veterans Affairs Merit Review (to K.E.D.); a gift from Malcolm E. and Musette Powell (to J.A.K.); and Training Grant T32-EY07092 (to H.P.L.) from the National Eye Institute, National Institutes of Health. J.A.K. is the recipient of the Jules and Doris Stein Research to Prevent Blindness Professorship of Ophthalmology awarded by Research to Prevent Blindness.

² Current address: Department of Pathology, Weill Medical College, Cornell University, New York, NY 10021.

³ Address correspondence and reprint requests to Dr. Judith A. Kapp, Emory Eye Center, 1365-B Clifton Road NE, Atlanta, GA 30322. E-mail address: jkapp{at}emory.edu

⁴ Abbreviations used in this paper: P2, purinergic; ATPase, adenosine triphosphatase; ADPase, adenosine diphosphatase; E-NTPDase, ecto-nucleoside triphosphate diphosphohydrolase; P_i, inorganic phosphate; ATPS, adenosine 5'-[-thio]triphosphatate; DMF, N,N'-dimethylformamide; 5'-FSBA, 5'-p-(fluorosulfonyl)benzoyl adenosine; E.G7-OVA, EL4 transfected with the OVA gene; SBA, sulfonylbenzoyladenosine.

Received for publication August 19, 2002. Accepted for publication January 6, 2003.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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