Memory of Extracellular Adenosine A2A Purinergic Receptor-mediated Signaling in Murine T Cells
1997; Elsevier BV; Volume: 272; Issue: 41 Linguagem: Inglês
10.1074/jbc.272.41.25881
ISSN1083-351X
AutoresM. Koshiba, Hidefumi Kojima, Steve Huang, Sergey Apasov, Michail V. Sitkovsky,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoAccumulation of extracellular and intracellular adenosine (Ado) under hypoxic conditions or in the absence of adenosine deaminase results in lymphocyte depletion and in severe combined immunodeficiency, which are currently explained by direct intracellular lymphotoxicity of Ado metabolites. In support of the alternative, “signaling” mechanism, we show that extracellular Ado (extAdo) suppresses all tested T cell receptor (TCR)-triggered effector functions of T lymphocytes including the TCR-triggered FasL mRNA up-regulation in cytotoxic T lymphocytes. Strong evidence against the intracellular lymphotoxicity of Ado (and in support of the signaling model) is provided by abrogation of TCR-triggered growth inhibition in Ado-exposed T cells. The brief exposure to Ado was sufficient to observe inhibition of TCR-triggered effector functions. The “memory” of T cells to exposure to extAdo is best explained by sustained increases in cAMP. Selective agonist (CGS21680) and antagonist (ZM241385) of A2A adenosine receptor were used in functional assays and cDNA probes for different sybtypes of adenosine receptors were used in Northern blot studies. A2Areceptors are identified as the predominantly expressed subtype of Gs-coupled Ado receptors in T cells. The demonstration of cross-talk between the A2A receptors and TCR in both directions support the possible role of A2A receptors in mechanisms of extAdo-mediated immunosuppression in vivounder adenosine deaminase deficiency and hypoxic conditions in,e.g., solid tumors. Accumulation of extracellular and intracellular adenosine (Ado) under hypoxic conditions or in the absence of adenosine deaminase results in lymphocyte depletion and in severe combined immunodeficiency, which are currently explained by direct intracellular lymphotoxicity of Ado metabolites. In support of the alternative, “signaling” mechanism, we show that extracellular Ado (extAdo) suppresses all tested T cell receptor (TCR)-triggered effector functions of T lymphocytes including the TCR-triggered FasL mRNA up-regulation in cytotoxic T lymphocytes. Strong evidence against the intracellular lymphotoxicity of Ado (and in support of the signaling model) is provided by abrogation of TCR-triggered growth inhibition in Ado-exposed T cells. The brief exposure to Ado was sufficient to observe inhibition of TCR-triggered effector functions. The “memory” of T cells to exposure to extAdo is best explained by sustained increases in cAMP. Selective agonist (CGS21680) and antagonist (ZM241385) of A2A adenosine receptor were used in functional assays and cDNA probes for different sybtypes of adenosine receptors were used in Northern blot studies. A2Areceptors are identified as the predominantly expressed subtype of Gs-coupled Ado receptors in T cells. The demonstration of cross-talk between the A2A receptors and TCR in both directions support the possible role of A2A receptors in mechanisms of extAdo-mediated immunosuppression in vivounder adenosine deaminase deficiency and hypoxic conditions in,e.g., solid tumors. Adenosine (Ado) 1The abbreviations used are: Ado, adenosine; 2-CADO, 2-chloroadenosine; ADA, adenosine deaminase; BLT, benzyloxycarbonyl-l-lysine thiobenzyl ester; CSC, 8-(3-chlorostiryl) caffeine; CTL, cytotoxic T lymphocyte; dbcAMP,N-6,2-O-dibutyryladenosine 3′,5′-cyclic monophosphate; ELISA, enzyme-linked immunosorbent assay; extAdo, extracellular adenosine; FasL, Fas ligand; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; mAb, monoclonal antibody; NECA, 5-N-ethylcarboxamide adenosine; ORF, open reading frame; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; SCID, severe combined immunodeficiency; TCR, T cell receptor. 1The abbreviations used are: Ado, adenosine; 2-CADO, 2-chloroadenosine; ADA, adenosine deaminase; BLT, benzyloxycarbonyl-l-lysine thiobenzyl ester; CSC, 8-(3-chlorostiryl) caffeine; CTL, cytotoxic T lymphocyte; dbcAMP,N-6,2-O-dibutyryladenosine 3′,5′-cyclic monophosphate; ELISA, enzyme-linked immunosorbent assay; extAdo, extracellular adenosine; FasL, Fas ligand; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; mAb, monoclonal antibody; NECA, 5-N-ethylcarboxamide adenosine; ORF, open reading frame; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; SCID, severe combined immunodeficiency; TCR, T cell receptor. has been implicated in pathogenesis of diseases (1Giblett E.R. Anderson J.E. Cohen F. Pollara B. Meuwissen H.J. Lancet. 1972; 2: 1067-1069Abstract PubMed Scopus (1158) Google Scholar, 2Hershfield M.S. Mitchell B.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Molecular and Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 1725-1768Google Scholar, 3Hirschhorn R. Clin. Immunol. Immunopathol. 1995; 76: S219-S227Crossref PubMed Scopus (52) Google Scholar, 4Blackburn M.R. Datta S.K. Wakamiya M. Vartabedian B.S. Kellems R.E. J. Biol. Chem. 1996; 271: 15203-15210Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 5Huang S. Koshiba M. Apasov S. Sitkovsky M. Blood. 1997; 90: 1600-1610Crossref PubMed Google Scholar) and in normal T cell immunity (6Apasov S. Koshiba M. Redegeld F. Sitkovsky M. Immunol. Rev. 1995; 146: 5-19Crossref PubMed Scopus (80) Google Scholar, 7Apasov S.G. Koshiba M. Chused T.M. Sitkovsky M.V. J. Immunol. 1997; 158: 5095-5105PubMed Google Scholar) and is considered to be an endogenous anti-inflammatory agent (8Cronstein B.N. Naime D. Firestein G. Arthritis Rheum. 1995; 38: 1040-1045Crossref PubMed Scopus (121) Google Scholar). Ado analogs are also used as pharmacological agents (9Olah M.E. Stiles G.L. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 581-606Crossref PubMed Google Scholar, 10Mizumura T. Auchampach J.A. Linden J. Bruns R.F. Gross G.J. Circ. Res. 1996; 79: 415-423Crossref PubMed Scopus (84) Google Scholar, 11Jacobson K.A. van Galen P.J.M. Williams M. J. Med. Chem. 1992; 35: 407-422Crossref PubMed Scopus (501) Google Scholar). Increased concentrations of Ado have been detected in the absence of adenosine deaminase activity (ADA) (4Blackburn M.R. Datta S.K. Wakamiya M. Vartabedian B.S. Kellems R.E. J. Biol. Chem. 1996; 271: 15203-15210Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) or under hypoxic conditions (e.g. large solid tumors) (12Hoskin D.W. Reynolds T. Blay J. Int. J. Cancer. 1994; 59: 854-855Crossref PubMed Scopus (69) Google Scholar). Accumulated Ado is believed to be lymphotoxic, and humans with inherited ADA deficiency (undetectable ADA activity) are ill and have severe combined immunodeficiency (SCID) (2Hershfield M.S. Mitchell B.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Molecular and Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 1725-1768Google Scholar, 3Hirschhorn R. Clin. Immunol. Immunopathol. 1995; 76: S219-S227Crossref PubMed Scopus (52) Google Scholar, 4Blackburn M.R. Datta S.K. Wakamiya M. Vartabedian B.S. Kellems R.E. J. Biol. Chem. 1996; 271: 15203-15210Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). ADA SCID is currently explained by the accumulation of lymphotoxic intracellular deoxyATP (dATP) and/or of S-adenosyl homocysteine as the cause of direct lymphotoxicity leading to the depletion of lymphocytes (2Hershfield M.S. Mitchell B.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Molecular and Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 1725-1768Google Scholar, 3Hirschhorn R. Clin. Immunol. Immunopathol. 1995; 76: S219-S227Crossref PubMed Scopus (52) Google Scholar, 4Blackburn M.R. Datta S.K. Wakamiya M. Vartabedian B.S. Kellems R.E. J. Biol. Chem. 1996; 271: 15203-15210Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 13Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4405) Google Scholar). Alternative mechanism(s) of effects of Ado on lymphocytes, as well as the possibility that their inhibition may be due to extracellular Ado-mediated transmembrane signaling through Ado (purinergic) receptors (5Huang S. Koshiba M. Apasov S. Sitkovsky M. Blood. 1997; 90: 1600-1610Crossref PubMed Google Scholar, 6Apasov S. Koshiba M. Redegeld F. Sitkovsky M. Immunol. Rev. 1995; 146: 5-19Crossref PubMed Scopus (80) Google Scholar, 14Kizaki H. Shimada H. Ohsaka F. Sakurada T. J. Immunol. 1988; 141: 1652-1657PubMed Google Scholar, 15van der Ploeg I. Ahlberg S. Parkinson F.E. Olsson R.A. Fredholm B.B. Naunyn Schmiedebergs Arch. Pharmacol. 1996; 353: 250-260Crossref PubMed Scopus (54) Google Scholar, 16McConkey D.J. Orrenius S. Jondal M. J. Immunol. 1990; 145: 1227-1230PubMed Google Scholar) on thymocytes and peripheral T cells, have not yet been carefully explored. According to this “signaling” model (5Huang S. Koshiba M. Apasov S. Sitkovsky M. Blood. 1997; 90: 1600-1610Crossref PubMed Google Scholar, 6Apasov S. Koshiba M. Redegeld F. Sitkovsky M. Immunol. Rev. 1995; 146: 5-19Crossref PubMed Scopus (80) Google Scholar), the accumulation of extAdo due to insufficient or absent ADA or under hypoxic conditions results in increased signaling through Ado receptors on T cells and causes immunosuppression. This signaling mechanism is attractive inasmuch as, if correct, it could provide a novel molecular target for immunomodulation; however, studies about the effect(s) of extAdo-triggered signaling on the effector functions of T cells and about the repertoire of expressed Ado receptors on T cells need to be performed. Clarification of the cellular and biochemical mechanisms of ADA SCID is important for the understanding of the pathogenesis of this disease. In addition, studies of the effects of extAdo on T cells are valuable in devising strategies based on the use of adoptively transferred cytotoxic T lymphocytes (CTLs) to patients with solid tumors where hypoxic conditions may lead to Ado accumulation (12Hoskin D.W. Reynolds T. Blay J. Int. J. Cancer. 1994; 59: 854-855Crossref PubMed Scopus (69) Google Scholar). Rational predictions of the side effects of Ado-based pharmacological agents also require this knowledge. Recent advances in understanding of mechanisms of T cell-mediated effector functions, together with the availability of molecular tools to study Ado receptors, will allow more detailed and precise evaluation of the effects of extAdo under well controlled conditions. In this study, we asked (i) whether Ado-induced immunosuppression of peripheral T lymphocytes in vivo may be at least partially explained by extracellular versus intracellular Ado-mediated inhibition of antigen/T cell receptor (TCR)-driven, perforin- and Fas-based, T lymphocyte-mediated cytotoxicity and lymphokine secretion and (ii) whether the effects of Ado could be explained by extAdo receptor-mediated signaling. Here we present data that demonstrate that even short term exposure of T cells to extAdo results in strong inhibition of TCR-triggered functions, thereby providing evidence of a “memory” of Ado effects in surrounding T cells. The ability of extAdo to rescue T cells from apoptosis strongly supports signalingversus an intracellular toxicity mechanism of Ado action. TCR-signaling antagonizing A2A receptors are identified as predominantly expressed and are probably functional transducers of the T cell-inhibiting effects of extAdo. All the reagents were purchased from Sigma unless otherwise mentioned. Selective A2A receptor agonist CGS21680 and selective A2A receptor antagonist CSC were purchased from RBI (Natick, MA). Selective A2A receptor antagonist ZM241385 was a kind gift from Dr. Joel Linden (University of Virginia, Charlottesville, VA) and Dr. Kenneth Jacobson (NIH, Bethesda, MD). Anti-CD3 ε-chain monoclonal antibody (mAb) 145-2C11 (2C11) was prepared as described (17Leo O. Foo M. Sachs D.H. Samelson L.E. Bluestone J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1374-1378Crossref PubMed Scopus (1697) Google Scholar). Anti-Thy-1 mAb (anti-Thy-1.2, clone 53-2.1) and anti-Fas ligand (FasL) mAb were obtained from PharMingen (San Diego, CA) and Alexis (San Diego, CA), respectively. Splenocytes and thymocytes were isolated from 4–6-week-old DBA/2 mice (H-2d). Mouse CD8+ CTL clone OE4 (anti-H-2d) (18Staerz U.D. Kanagawa O. Bevan M.J. Nature. 1985; 314: 628-631Crossref PubMed Scopus (375) Google Scholar) was stimulated every 4 weeks with irradiated splenocytes from DBA/2 mice in EL4-conditioned medium, expanded 4 days after stimulation, and harvested at day 8 (4 days after expansion) (19Takayama H. Shinohara N. Kawasaki A. Someya Y. Hanaoka S. Kojima H. Yagita H. Okumura K. Shinkai Y. Int. Immunol. 1991; 3: 1149-1156Crossref PubMed Scopus (35) Google Scholar). Mouse B cell lymphoma line A20.2J (H-2d) and mouse T cell leukemia line EL4 (H-2b) were used as specific and nonspecific target cells for OE4, respectively. The perforin-deficient, FasL-expressing CD8+ CTL line P0K.3A8 (anti-H-2k) was maintained as described previously (20Kojima H. Shinohara N. Hanaoka S. Someya-Shirota Y. Takagaki Y. Ohno H. Saitoh T. Katayama T. Yagita H. Okumura K. Shinkai Y. Alt F.W. Matsuzawa A. Yonehara S. Takayama H. Immunity. 1994; 1: 357-364Abstract Full Text PDF PubMed Scopus (282) Google Scholar). The perforin-deficient, FasL-expressing CD4+ CTL line P0P.C 2H. Kojima, unpublished results. was maintained by biweekly stimulation and expansion with irradiated spleen cells. They were usually used 12 days after the last stimulation and were cultured in complete RPMI medium supplemented with 10% FCS and 2.5% supernatant from concanavalin A-stimulated rat spleen cells as source of T cell growth factors. B cell hybridoma LB 27.4 (LB; H-2b/d) (21Kappler J. White J. Wegmann D. Mustain E. Marrack P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3604-3607Crossref PubMed Scopus (210) Google Scholar) was sensitive to Fas-mediated cytotoxicity and used as a target of CTL P0P.C. Mouse T helper hybridoma 2B4.11 (2B4) was prepared as described previously (22Sugiyama H. Chen P. Hunter M. Taffs R. Sitkovsky M. J. Biol. Chem. 1992; 267: 25256-25263Abstract Full Text PDF PubMed Google Scholar). Secreted benzyloxycarbonyl-l-lysine thiobenzyl ester (BLT; Calbiochem) esterase activity was measured as described previously (23Takayama H. Sitkovsky M.V. J. Exp. Med. 1987; 166: 725-743Crossref PubMed Scopus (106) Google Scholar). Typically, the amount of secreted BLT esterase was determined after the incubation of 1 × 105 CTLs in wells coated with immobilized 2C11 mAb (5 μg/ml) with or without Ado analogs. After 4 h of incubation at 37 °C under 5% CO2, cells were centrifuged at 200 × g for 5 min. To assay BLT esterase activity, 50 μl of culture supernatant was mixed with 950 μl of BLT solution (0.2 mm BLT, 0.22 mm5,5′-dithio-bis-(2-nitrobenzoic acid) (Calbiochem) in phosphate-buffered saline, pH 7.2). The mixture was incubated at 37 °C for 20 min, and the reaction was stopped by adding 10 μl of 0.1 m phenylmethanesulfonyl fluoride, which was dissolved in Me2SO. The solution was diluted by 1.0 ml of phosphate-buffered saline, and the absorbance at 412 nm was measured in comparison to a blank solution (RPMI 1640, 10 mm HEPES, 5% FCS) that was treated exactly the same as the experimental solution. Inasmuch as the culture medium does not react with 5,5′-dithio-bis-(2-nitrobenzoic acid), the experimental absorbance value minus the blank absorbance value is proportional to the BLT esterase activity. The total cellular content of the enzyme was determined using 0.1% Triton X-100-solubilized cells. Determinations were carried out in triplicates, and data are presented as the specific percent of enzymatic activity released, which was calculated from the following equation. %release=100×(E−S)/(T−S)Equation 1 E represents the mean absorbance value in the supernatants of the experimental wells, S is the mean absorbance value in the supernatants of the wells containing no stimuli, and T represents the total enzyme content of BLT esterase in the CTLs. CTL cytotoxicity was measured using a standard 51Cr release assay (24Kataoka T. Shinohara N. Takayama H. Takaku K. Kondo S. Yonehara S. Nagai K. J. Immunol. 1996; 156: 3678-3686PubMed Google Scholar). Briefly, target cells were labeled with 50 μCi of Na251CrO4 for 1 h at 37 °C and then washed three times. 51Cr-Labeled target cells (2.5 × 103) were incubated with different numbers of CTLs as indicated according to effector/target (E:T) ratio. With or without Ado or Ado analogs in 200 μl of complete RPMI 1640 medium with 5% FCS, cells were incubated for 4 h at 37 °C in V-bottomed 96-well plates. Culture supernatants were harvested using the Skatron harvesting system (Skatron, Lier, Norway). Cytolytic activity, i.e. percentage of specific 51Cr release, was calculated as follows. %of specific 51Cr release =100 × (E−S)/(T−S)Equation 2 E, S, and T indicate experimental release, spontaneous release, and total incorporation, respectively. In this assay, the A20.2J cells (H-2d) were used as targets. These cells are normally not recognized and therefore not killed by P0K.3A8 CTLs (anti-H-2k); however, the binding of anti-CD3 mAb to Fc receptors on A20.2J cells results in CTL/A20.2J conjugation and TCR triggering of the CTLs, which is followed by activation of the CTLs and the lethal hit delivery to targets, so that the target cells become susceptible to lysis (25Taffs R.E. Redegeld F. Sitkovsky M.V. J. Immunol. 1991; 147: 722-729PubMed Google Scholar, 26Ratner A. Clark W. Sitkovsky M.V. Henkart P.A. Cytotoxic Cells: Recognition, Effector Functions, Generation, and Methods. Birkhäuser, Boston, 1993: 487Google Scholar). 1 × 105 OE4 cells/sample were incubated with plate-bound anti-CD3 mAb 2C11 (5 μg/ml) alone or together with 50 μm extAdo with or without 1 μm ZM241385. Total RNA was prepared by the single-step method of Chomczynski and Sacchi (RNA STAT-60; Tel-Test “B”, Friendswood, TX). After DNase I treatment the first-strand cDNA was synthesized by the SuperScript preamplification system (Life Technologies, Inc.) according to the manufacturer's instructions. The mouse FasL sequence was amplified with primers corresponding to 9–28 and 741–722 nucleotides of open reading frame (ORF) of mouse FasL cDNA (27Suda T. Takahashi T. Golstein P. Nagata S. Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2429) Google Scholar). All PCR primers used were synthesized by Genosys (The Woodlands, TX). The double-strand competitor DNA for quantitation of FasL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA by RT-PCR were prepared using the PCR MIMIC Construction Kit (CLONTECH Laboratories, Palo Alto, CA) according to the manufacturer's instructions. The amplification efficiency of each competitor sequence did not show any significant difference from that of the corresponding target cDNA sequence (data not shown). The competitive PCR was performed under standard conditions in a 30-μl reaction volume that included 3 μl of the diluted cDNA and 1.2 μl of competitor DNA. Ten μl of PCR product were electrophoresed on a 4% (w/v) NuSieve 3:1 agarose gel (FMC BioProducts, Rockland, ME) and examined by ethidium bromide staining. The density of each band was determined (ImageQuant version 3.3, Molecular Dynamics, Sunnyvale, CA) from the negative picture of the gel (no. 55 positive-negative 4 × 5 instant sheet film, Polaroid, Cambridge, MA). The densities of RT-PCR products were normalized for differences in cDNA quantity between samples using PCR quantitation of GAPDH mRNA. Primers for mouse GAPDH cDNA amplification were 290–309 and 1010–989 nucleotides of ORF. The total RNA from murine lymphoid cells was extracted as above and poly(A) RNA was subsequently isolated using Oligotex mRNA Mini Kit (Qiagen, Chatsworth, CA). The poly(A) RNA from 250 μg of total RNA was analyzed by standard Northern blotting procedure described previously (5Huang S. Koshiba M. Apasov S. Sitkovsky M. Blood. 1997; 90: 1600-1610Crossref PubMed Google Scholar). The first-strand cDNA was prepared from OE4 and 2B4 cells as described above. The mouse A2A sequence was amplified with primers corresponding to 556–575 and 927–910 nucleotides of ORF of mouse A2A cDNA (28Marquardt D.L. Walker L.L. Heinemann S. J. Immunol. 1994; 152: 4508-4515PubMed Google Scholar). A2A cDNA was a kind gift from Dr. D. L. Marquardt (University of California, San Diego) and used as a positive control. The effects of different treatments of lymphocytes on their cAMP content were tested by incubating 0.5–1.0 × 106 cells alone or with different combinations of Ado analogs and/or immobilized antibodies to CD3 or Thy-1 surface antigens. Levels of cAMP were measured using the cAMP EIA System (Amersham) according to the manufacturer's protocol. Measurements were done in triplicate with standard deviations calculated. The effect of extAdo pretreatment on anti-CD3 mAb (2C11)-induced CTL granule exocytosis was tested by preincubating cells for 30, 20, 10, 5, 1, or 0 min with 50 μm 2-CADO in 5% FCS-containing medium. Cells were washed and incubated for 4 h in triplicate in a V-bottomed 96-well plate with 2C11 mAb immobilized at 5 μg/ml, and granule exocytosis was calculated as described. Effect of 2-CADO pretreatment on anti-CD3 mAb (2C11)-induced cell growth inhibition and IL-2 production of 2B4 cells (22Sugiyama H. Chen P. Hunter M. Taffs R. Sitkovsky M. J. Biol. Chem. 1992; 267: 25256-25263Abstract Full Text PDF PubMed Google Scholar) was tested by preincubating cells for 30, 20, 10, 5, 1, or 0 min with 50 μm 2-CADO in 5% FCS-containing medium in triplicate. Then cells were washed, incubated for 21 h with immobilized 2C11, and pulsed with 1 μCi of [3H]thymidine, followed by an additional 6-h incubation at 37 °C, 5% CO2 in the presence of 2C11. The amount of secreted IL-2 in supernatants of 2B4 was estimated by sandwich ELISA (Intertest-2X mouse IL-2 ELISA kit; Genzyme, Cambridge, MA). CTL-mediated cytotoxicity involves several steps including early biochemical events of TCR-mediated signaling, conjugate formation, and lethal hit delivery (29Martz E. Sitkovsky M. Henkart P. Cytotoxic Cells: Recognition, Effector Functions, Generation, and Methods. Birkhäuser, Boston, 1993: 9-49Google Scholar). CTL-mediated cytotoxicity is explained by two complementary mechanisms. CTLs kill target cells (i) due to the exocytosis of perforin- and granzyme-containing cytotoxic granules (30Henkart P.A. Hayes M.P. Shiver J.W. Sitkovsky M.V. Henkart P.A. Cytotoxic Cells: Recognition, Effector Function, Generation, and Methods. Birkhäuser, Boston, 1992: 153-165Google Scholar) and (ii) due to FasL/Fas receptor interactions (31Rouvier E. Luciani M.-F. Golstein P. J. Exp. Med. 1993; 177: 195-200Crossref PubMed Scopus (802) Google Scholar). These are two completely different mechanisms that have in common only the requirement for TCR triggering. It was important to determine which of these pathways (or both) was susceptible to effects of extAdo. Different combinations of effector CTLs and target cells and the use of a granule exocytosis assay allowed us to use genetic controls to discriminate between Fas-mediated and granule exocytosis-based cytotoxicity. We demonstrated earlier that [cAMP] i-elevating treatments of CTLs result in inhibition of both early and late biochemical events leading to CTL-mediated cytotoxicity and to exocytosis of cytolytic granules (32Takayama H. Trenn G. Sitkovsky M.V. J. Biol. Chem. 1988; 263: 2330-2336Abstract Full Text PDF PubMed Google Scholar). However, in our earlier model studies, we did not use relatively low intensity extracellular signaling as a method of raising cAMP levels. Therefore, it was interesting to test whether extAdo-induced levels of cAMP will be sufficient to cause the inhibition of CTL functions if signaling through Ado receptors in peripheral T cells involves Gsprotein-coupled and cAMP-raising A2A or the A2Bsubtypes of receptors (11Jacobson K.A. van Galen P.J.M. Williams M. J. Med. Chem. 1992; 35: 407-422Crossref PubMed Scopus (501) Google Scholar). We found that the presence of Ado analogs significantly inhibits TCR-triggered granule enzyme secretion (Fig.1 A), thereby providing the first demonstration of direct effects of extAdo analogs on cytotoxicity-mediating granule exocytosis in CTLs. As little as 1.0 μm NECA was able to inhibit granule exocytosis (Fig.1 A), and both NECA and 2-CADO were efficient in inhibition. Inhibition of exocytosis of cytotoxic granules in CTLs by Ado (Fig.1 A) was expected to result in the inhibition of target cell lysis, and this was demonstrated by testing the effect of extAdo on target cell lysis by CTLs (Fig. 1 B). The CTL clone used was unable to lyse the nonspecific targets, as demonstrated by the control experiment (Fig. 1 B, inset), thereby confirming that target cell lysis is antigen-specific and TCR-triggered. It was found that inclusion of Ado in the assay significantly inhibited target cell death. Of interest, not only Ado but also Ado analogs (2-CADO, NECA) were inhibitory, and up to 500 μm Ado was as efficient as nonhydrolyzable 2-CADO and NECA, suggesting that signaling, rather than Ado degradation (metabolism) products, is responsible for these effects. Selective A2A agonist CGS21680 at concentrations as low as 0.1 μm (Fig.1 B, lower left panel) also was able to inhibit the lysis of antigen-specific target cells (A20.2J) by CTL clone OE4. At such low concentrations, this agonist was shown to be highly selective for A2A receptor (33Hutchinson A.J. Williams M. de Jesus R. Yokoyama R. Oei H.H. Ghai G.R. Webb R.L. Zoganas H.C. Stone G.A. Jarvis M.F. J. Med. Chem. 1990; 33: 1919-1924Crossref PubMed Scopus (114) Google Scholar). The degree of inhibition by CGS21680 was comparable to that of extracellular adenosine and of 2-CADO even at higher concentrations of 50 μm (Fig.1 B, lower right panel). The most selective to date (34Poucher S.M. Keddie J.R. Singh P. Stoggall S.M. Caulkett P.W.R. Jones G. Collis M.G. Br. J. Pharmacol. 1995; 115: 1096-1102Crossref PubMed Scopus (324) Google Scholar) A2A adenosine receptor antagonist ZM241385 was also used to identify the A2Areceptor in mediating effects of adenosine on T cells. It is shown (Fig. 1 A, lower panel) that the presence of ZM241385 at 1 μm was sufficient to almost completely protect CTL even from the exposure to the highest tested (50 μm) concentration of 2-CADO. In a control experiment, ZM241385 alone at 1 μm did not have an effect on exocytosis of TCR-triggered CTL. Thus, both antigen receptor-triggered exocytosis of cytotoxic granules and specific antigen-triggered T cell lysis were inhibited by extAdo. Although the effects of extAdo on processes in target cells undergoing perforin-induced death are still to be established, these data allow us to conclude that granule exocytosis-based CTL-mediated cytotoxicity is inhibited by extAdo. The inhibition of exocytosis was highly reproducible, but the degree of inhibition varied depending on the extent of TCR-triggered exocytosis in the particular assay and on the preparation of expanded CTL clones. Both nonhydrolyzable analogs 2-CADO and NECA were effective in inhibition of the protein synthesis-independent short term process of granule exocytosis, suggesting that these effects are due to signaling rather than to the interference into DNA synthetic pathways and dATP-triggered apoptosis (13Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4405) Google Scholar). Control experiments were performed to consider the possibility that the decrease in CTL viability by Ado and anti-CD3 mAb may account for the Ado-mediated inhibition of granule exocytosis and cytotoxicity. In controls, no changes in viability of CTLs were detected using trypan blue (data not shown). In addition, this possibility is discounted by results of flow cytometry (forward scatter versus side scatter) measurements of CTL viability, inasmuch as even at very high concentrations (e.g. 5 mm) of extAdo more than 70% of CTLs were viable and not apoptotic. To study the susceptibility of Fas-versus perforin-mediated and FasL/Fas cytotoxicity pathways to inhibition by Ado, we took advantage of the availability of Fas-expressing and Fas-mediated death pathway-sensitive targets and tested the lysis of such targets with a perforin-deficient CTLs that possesses only the FasL-based mechanism of cytotoxicity. The CD4+ CTL line P0P.C cells used in the experiment described in Fig.2 A were derived from perforin-deficient mice; thus, the cytotoxicity of these CTLs depends only on the Fas-mediated pathway. It is shown (Fig. 2 A) that extAdo and Ado analog 2-CADO were able to inhibit the cytotoxicity of P0P.C CTLs against the antigen-bearing target cells in a dose-dependent manner. Inhibition by extAdo was observed at as low as 100 μm Ado, with poorly hydrolyzable analog 2-CADO being much more efficient in inhibition of Fas-mediated cytotoxicity of CTLs. This inhibition of Fas-mediated cytotoxicity could be due to (i) inhibition of adhesion protein-mediated CTL/target conjugate formation, (ii) the interference of extAdo into intracellular processes of cell death in targets, and/or (iii) inhibition of TCR-triggered FasL up-regulation. The latter was the most interesting explanation of extAdo-mediated inhibition of FasL-triggered cell death in CTL cytotoxicity assays, but it was necessary to eliminate from considerations the contribution of CTL/target conjugate formation and programmed cell death processes observed in inhibition of Fas-mediated cytotoxicity (Fig. 2 A). To exclude the possibility of inhibition of CTL/target cell conjugate formation, we utilized a “retargeting” assay in which the target cells were “armed” with anti-TCR/CD3 complex mAb. In this assay, the target cell “recognizes” the TCR on the CTL surface, and antibody/TCR links alone are s
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