Proteome Analysis Reveals Caspase Activation in Hyporesponsive CD4 T Lymphocytes Induced in Vivo by the Oral Administration of Antigen
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m212820200
ISSN1083-351X
AutoresTomohiro Kaji, Satoshi Hachimura, Wataru Ise, Shuichi Kaminogawa,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThe oral administration of antigen can lead to systemic antigen-specific hyporesponsiveness, also known as oral tolerance. This phenomenon is a representative form of immune tolerance to exogenous antigen under physiological conditions. We have previously reported that long term feeding of dietary antigen to ovalbumin-specific T cell receptor (TCR) transgenic mice induced oral tolerance of peripheral T cells with impairment in their TCR-induced calcium-signaling pathway. In this study, we utilized two-dimensional electrophoresis to compare intracellular protein expression patterns of orally tolerant and unsensitized CD4 T cells. We detected 26 increased and 16 decreased protein spots and identified 35 of these by mass spectrometry. The results indicated that the expression of caspases was up-regulated and that the protein levels of intact proteins susceptible to caspase cleavage, such as Grb2-related adaptor downstream of Shc (GADS), were decreased in orally tolerant CD4 T cells. Western blotting experiments confirmed that expression of the active form of caspase-3 and the antiapoptotic factor, X-linked inhibitor of apoptosis, were both up-regulated in orally tolerant CD4 T cells, which were found to be nonapoptotic. We further demonstrated that orally tolerant CD4 T cells could not form normal TCR signaling complexes associated with GADS and showed down-regulated phospholipase C-γ1 activation, which is likely to contribute to the impairment of TCR-induced calcium signaling. Our findings indicate that orally tolerant CD4 T cells up-regulate caspase activation and show decreased levels of caspase-targeted proteins, including TCR signaling-associated molecules, while up-regulating antiapoptotic factors, all of which appear to contribute to their unique tolerant characteristics. The oral administration of antigen can lead to systemic antigen-specific hyporesponsiveness, also known as oral tolerance. This phenomenon is a representative form of immune tolerance to exogenous antigen under physiological conditions. We have previously reported that long term feeding of dietary antigen to ovalbumin-specific T cell receptor (TCR) transgenic mice induced oral tolerance of peripheral T cells with impairment in their TCR-induced calcium-signaling pathway. In this study, we utilized two-dimensional electrophoresis to compare intracellular protein expression patterns of orally tolerant and unsensitized CD4 T cells. We detected 26 increased and 16 decreased protein spots and identified 35 of these by mass spectrometry. The results indicated that the expression of caspases was up-regulated and that the protein levels of intact proteins susceptible to caspase cleavage, such as Grb2-related adaptor downstream of Shc (GADS), were decreased in orally tolerant CD4 T cells. Western blotting experiments confirmed that expression of the active form of caspase-3 and the antiapoptotic factor, X-linked inhibitor of apoptosis, were both up-regulated in orally tolerant CD4 T cells, which were found to be nonapoptotic. We further demonstrated that orally tolerant CD4 T cells could not form normal TCR signaling complexes associated with GADS and showed down-regulated phospholipase C-γ1 activation, which is likely to contribute to the impairment of TCR-induced calcium signaling. Our findings indicate that orally tolerant CD4 T cells up-regulate caspase activation and show decreased levels of caspase-targeted proteins, including TCR signaling-associated molecules, while up-regulating antiapoptotic factors, all of which appear to contribute to their unique tolerant characteristics. The oral administration of antigen (Ag) 1The abbreviations used are: Ag, antigen; IL, interleukin; NFAT, nuclear factor of activated T cells; OVA, ovalbumin; TCR, T cell receptor; 2-DE, two-dimensional electrophoresis; MS, mass spectrometry; Ab, antibody; mAb, monoclonal antibody; pAb, polyclonal antibody; PI, propidium iodide; XIAP, X-linked inhibitor of apoptosis; GADS, Grb2-related adaptor downstream of Shc; SLP-76, Src homology 2 domain-containing leukocyte protein of 76 kDa; LAT, linker for activation of T cells; PLC-γ1, phospholipase C-γ1; APC, antigen-presenting cell; IFN-γ, interferon-γ; IAP, inhibitor of apoptosis; FITC, fluorescein isothiocyanate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight. can lead to systemic Ag-specific hyporesponsiveness, also known as oral tolerance. This phenomenon is thought to prevent hypersensitivity to ingested antigen (1Strober W. Kelsall B. Marth T. J. Clin. Immunol. 1998; 18: 1-30Crossref PubMed Scopus (133) Google Scholar, 2Garside P. Mowat A.M. Semin. Immunol. 2001; 13: 177-185Crossref PubMed Scopus (118) Google Scholar) and is a representative form of peripheral tolerance to exogenous Ag under physiological conditions (3Strobel S. Ann. N. Y. Acad. Sci. 2002; 958: 47-58Crossref PubMed Scopus (52) Google Scholar). In addition, it is believed to be an effective therapeutic tool for the treatment of autoimmune diseases (4Faria A.M. Weiner H.L. Adv. Immunol. 1999; 73: 153-264Crossref PubMed Google Scholar, 5Hanninen A. Scand. J. Immunol. 2000; 52: 217-225Crossref PubMed Scopus (18) Google Scholar). As in the case of other forms of tolerance, oral tolerance is mediated at the T cell level (6Hirahara K. Hisatsune T. Nishijima K. Kato H. Shiho O. Kaminogawa S. J. Immunol. 1995; 154: 6238-6245PubMed Google Scholar, 7Alpan O. Rudomen G. Matzinger P. J. Immunol. 2001; 166: 4843-4852Crossref PubMed Scopus (142) Google Scholar). Three mechanisms are well documented for oral tolerance induction: (a) Ag-specific T cell clonal deletion (8Chen Y. Inobe J. Marks R. Gonnella P. Kuchroo V.K. Weiner H.L. Nature. 1995; 376: 177-180Crossref PubMed Scopus (738) Google Scholar, 9Garside P. Steel M. Worthey E.A. Kewin P.J. Howie S.E. Harrison D.J. Bishop D. Mowat A.M. Am. J. Pathol. 1996; 149: 1971-1979PubMed Google Scholar), (b) functional unresponsiveness of T cells to Ags (anergy) (10Whitacre C.C. Gienapp I.E. Orosz C.G. Bitar D.M. J. Immunol. 1991; 147: 2155-2163PubMed Google Scholar, 11Melamed D. Friedman A. Eur. J. Immunol. 1993; 23: 935-942Crossref PubMed Scopus (220) Google Scholar, 12Migita K. Ochi A. Eur. J. Immunol. 1994; 24: 2081-2086Crossref PubMed Scopus (30) Google Scholar, 13Van Houten N. Blake S.F. J. 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Previous studies demonstrated that Ag-specific T cell hyporesponsiveness was caused by defects in intracellular signaling events from the T cell receptor (TCR) and co-stimulatory molecules, such as incomplete protein phosphorylation (18Sloan-Lancaster J. Shaw A.S. Rothbard J.B. Allen P.M. Cell. 1994; 79: 913-922Abstract Full Text PDF PubMed Scopus (586) Google Scholar, 19Dubois P.M. Andris F. Shapiro R.A. Gilliland L.K. Kaufman M. Urbain J. Ledbetter J.A. Leo O. Eur. J. Immunol. 1994; 24: 348-354Crossref PubMed Scopus (17) Google Scholar, 20Becker J.C. Brabletz T. Kirchner T. Conrad C.T. Brocker E.B. Reisfeld R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2375-2378Crossref PubMed Scopus (68) Google Scholar, 21Migita K. Eguchi K. Kawabe Y. Tsukada T. Ichinose Y. Nagataki S. Ochi A. J. Immunol. 1995; 155: 5083-5087PubMed Google Scholar) and impaired calcium/nuclear factor of activated T cells (NFAT) signaling (22Kimura M. Yamashita M. Kubo M. Iwashima M. Shimizu C. Tokoyoda K. Chiba J. Taniguchi M. Katsumata M. Nakayama T. Int. Immunol. 2000; 12: 817-824Crossref PubMed Scopus (24) Google Scholar). Furthermore, genetic experiments, such as DNA array methods, have recently been performed to analyze hyporesponsive T cells entirely from multidirectional points (23Lechner O. Lauber J. Franzke A. Sarukhan A. von Boehmer H. Buer J. Curr. Biol. 2001; 11: 587-595Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Gavin M.A. Clarke S.R. Negrou E. Gallegos A. Rudensky A. Nat. Immunol. 2002; 3: 33-41Crossref PubMed Scopus (557) Google Scholar, 25Macian F. Garcia-Cozar F. Im S.H. Horton H.F. Byrne M.C. Rao A. Cell. 2002; 109: 719-731Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). However, to date, there are no detailed studies dealing with tolerant T cells examining total specific protein expression levels. In the postgenomic era, proteome analysis is expected to be the bridge between the genomic sequence and protein characteristics underlying cellular behavior, since translated proteins can be posttranslationally modified (26Anderson N.L. Anderson N.G. Electrophoresis. 1998; 19: 1853-1861Crossref PubMed Scopus (824) Google Scholar, 27Pandey A. Mann M. Nature. 2000; 405: 837-846Crossref PubMed Scopus (1977) Google Scholar, 28Mann M. Hendrickson R.C. Pandey A. Annu. Rev. Biochem. 2001; 70: 437-473Crossref PubMed Scopus (942) Google Scholar). The development of two-dimensional electrophoresis (2-DE) provides the high resolution of complex protein mixtures with high reproducibility. In addition, the combination of 2-DE, modern mass spectrometry (MS) technology, and rapidly accumulating genomic sequence data permits accurate and speedy identification of cellular proteins. With these advances, proteome analysis can be utilized in the research of intracellular protein changes in cells following a certain stimulus. We have previously reported that long term feeding of dietary Ag to ovalbumin (OVA)-specific TCR transgenic mice (OVA23-3 mice) induced oral tolerance of peripheral T cells (29Asai K. Hachimura S. Kimura M. Toraya T. Yamashita M. Nakayama T. Kaminogawa S. J. Immunol. 2002; 169: 4723-4731Crossref PubMed Scopus (39) Google Scholar). In this experimental model, orally tolerant T cells showed impaired calcium/NFAT signaling following TCR-mediated activation, despite normal activation of the mitogen-activated kinase pathway. In this study, we used oral tolerance-induced OVA23-3 mice to investigate the expression of intracellular proteins using 2-DE to analyze the specific characteristics of in vivo orally tolerized splenic CD4 T cells compared with unsensitized CD4 T cells. Mice—8–10-week-old BALB/c male mice were purchased from Clea Inc. (Tokyo, Japan). Mice homozygous for the OVA peptide323–339/I-Ad-restricted TCR transgene from the T cell clone 7-3-7 on the BALB/c background (OVA23-3 mice) were produced as previously described (30Sato T. Sasahara T. Nakamura Y. Osaki T. Hasegawa T. Tadakuma T. Arata Y. Kumagai Y. Katsuki M. Habu S. Eur. J. Immunol. 1994; 24: 1512-1516Crossref PubMed Scopus (94) Google Scholar). All studies were performed according to the Guidelines for Animal Experiments of the Faculty of Agriculture (University of Tokyo). OVA-specific Oral Tolerance Induction in OVA23-3 Mice—6–8-week-old OVA23-3 mice were orally administered with dietary Ag containing 20% hen egg white (egg white diet; Funabashi Farm, Funabashi, Japan) or a control diet (commercially available CE-2; Clea Inc.), for 28–30 days. Antibodies (Abs) and Reagents—For purification of CD4 T cells and flow cytometric analyses, FITC- or allophycocyanin-conjugated anti-mouse CD4 monoclonal Ab (mAb) (H129.19), biotinylated anti-mouse CD45R/B220 mAb (RA3-6B2), and phycoerythrin-conjugated anti-mouse TCRβ chain mAb (H57-597) were all purchased from BD PharMingen (San Diego, CA). Anti-mouse major histocompatibility complex class II mAb (M5/114.15.2) and anti-mouse CD11c mAb (N418) were purified from ascites and biotinylated in our laboratory according to standard techniques. Propidium iodide (PI) was purchased from Sigma. For the enrichment of CD4-positive cells, anti-mouse CD4 microbeads (Militenyi Biotec, Bergisch Gladbach, Germany) were used. For immunoprecipitation and Western blotting experiments, anti-X-linked inhibitor of apoptosis (XIAP) mAb (BD Transduction Laboratories, Lexington, KY), anti-Grb2-related adaptor downstream of Shc (GADS) polyclonal Ab (pAb) (Upstate Biotechnology, Inc., Lake Placid, NY), anti-phospholipase C-γ1 (PLC-γ1) mAb mixture (Upstate Biotechnology), anti-linker for activation of T cells (LAT) pAb (Upstate Biotechnology), anti-Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) pAb (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-TCR-ζ chain mAb (Zymed Laboratories Inc., South San Francisco, CA), horseradish peroxidase-conjugated anti-phosphotyrosine mAb (RC20; BD Transduction Laboratories), horseradish peroxidase-conjugated anti-rabbit IgG, and anti-mouse IgG (New England Biolabs, Boston, MA) were used. Preparation of Splenic CD4 T Lymphocytes—Preparation of splenic CD4 T cells of OVA23-3 mice was performed using an Anti-FITC MultiSort kit (Militenyi Biotec) as follows. Splenocytes were labeled with anti-mouse CD4 mAb conjugated with FITC, biotinylated anti-mouse major histocompatibility complex class II mAb, biotinylated anti-mouse CD11c mAb, and biotinylated anti-mouse CD45R/B220 mAb. After washing and secondary labeling with anti-FITC microbeads, cells were subjected to positive enrichment via the magnetic column. After release of the anti-FITC microbeads, cells were relabeled with streptavidin-conjugated microbeads for negative selection. Isolated cells were both CD4- and TCRβ chain-positive and at greater than 98% purity, as assessed by flow cytometric analyses, and were regarded as CD4 T lymphocytes. Proliferation Assay and Cytokine Assay with Enzyme-linked Immunosorbent Assay—1 × 105 purified CD4 T cells were cultured with 0, 0.1, or 1.0 mg/ml OVA in flat bottom 96-well plates and 4 × 105 mitomycin C-treated BALB/c splenocytes as APCs in RPMI 1640 medium containing 5% heat-inactivated fetal calf serum (Sigma). For cytokine assays, supernatant samples were collected at 24 h for IL-2 and 48 h for IL-4, interferon (IFN)-γ, and IL-10. For the proliferation assays, cultures were pulsed with [3H]thymidine (1 μCi/well, ICN Pharmaceuticals, Costa Mesa, CA) after 48 h of incubation and harvested 24 h later. Cells were collected on glass fiber, and the incorporated radioactivity was measured by scintillation counting. IL-2, IL-4, and IFN-γ cytokine production was measured by enzyme-linked immunosorbent assay using paired Abs (BD PharMingen), as described previously (31Yoshida T. Hachimura S. Kaminogawa S. Clin. Immunol. Immunopathol. 1997; 82: 207-215Crossref PubMed Scopus (46) Google Scholar). IL-10 production was measured using the OptEIA™ mouse IL-10 set (BD PharMingen) according to the manufacturer's instructions. 2-DE and Silver Staining—Purified CD4 T lymphocytes were lysed in 7 m urea, 2 m thiourea, 4% CHAPS, 20 mm dithiothreitol, 2.5 μg/ml DNase I, 2.5 μg/ml RNase, protease inhibitor mixture (Sigma), followed by sonication. Samples were then centrifuged at 20,000 × g for 1 h, and the supernatants were used as the whole cell lysates. The first dimension of 2-DE was performed with linear pH 4.5–5.5 and 5.5–6.7/18-cm Immobiline DryStrips (Amersham Biosciences). DryStrips were rehydrated overnight at room temperature in 8 m urea, 30 mm dithiothreitol, 2% CHAPS, 2% IPG buffer (Amersham Biosciences) containing 150–250 μg of proteins. The first dimension electrophoresis was run using the following conditions: 100 V for 3 h, 300 V for 1 h, 500 V for 2 h, 700 V for 1 h, 1,000 V for 1 h, 1,500 V for 1 h, 2,000 V for 1 h, 2,500 V for 1 h, and 3,000 V for 20–24 h. After the first run, Immobiline DryStrips were equilibrated according to the manufacturer's recommendations and set on the 12.0% polyacrylamide gels for the second dimensional separation. After 2-DE, gels were silver-stained as previously described (32Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7885) Google Scholar). Stained gels were imaged with Gel dock (Bio-Rad), and spot intensity was measured using ImageGauge software (Fuji Photo Film, Kanagawa, Japan). Normalized spot intensities were calculated from more than 10 spots of each gel. Protein Identification by MALDI-TOF MS—In-gel tryptic digestions of excised protein spots were performed with modified protocols as previously described (33Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (843) Google Scholar). Briefly, destained gels were reduced and alkylated, followed by tryptic digestion (Promega, Madison, WI). Digested peptides were eluted with 5% formic acid, 50% acetonitrile. Peptides were concentrated and desalted using ZipTipC18 (Millipore Corp., Bedford, MA) according to the manufacturer's protocol. Purified peptides were mixed with 10 mg/ml α-cyano-4-hydroxycinnamic acid, 0.2% aqueous trifluoroacetic acid, acetonitrile (1:1) as a matrix. The mass spectra were recorded by Voyager-DE STR (Applied Biosystems, Foster City, CA). Internal calibration was performed using autoproteolytic trypsin peptide fragments of 842.50 and 2211.1046 Da, and specific peaks were applied to the MS-fit program (available on the World Wide Web at prospector.ucsf.edu/ucsfhtml4.0/msfit.htm). Proteins were identified with 50 ppm accuracy and a minimum of four matching peptides and 25% peptide coverage of total amino acids. Stimulation of CD4 T Lymphocytes with Abs—Purified CD4 T cells were stimulated with co-cross-linking of TCR and CD4 molecules with Abs for the indicated times as previously described in Ref. 29Asai K. Hachimura S. Kimura M. Toraya T. Yamashita M. Nakayama T. Kaminogawa S. J. Immunol. 2002; 169: 4723-4731Crossref PubMed Scopus (39) Google Scholar. Immunoprecipitation and Western Blotting—For Western blots of the pro form and active form of caspase-3, harvested cells were lysed in CHAPS cell extract buffer (50 mm Pipes/KOH, pH 6.5, 0.1% CHAPS, 2 mm EDTA, 5 mm dithiothreitol, protease inhibitor), and detections were performed using an apoptosis sampler kit (Cell Signaling Technology, Beverly, MA) according to the manufacturer's instructions. Harvested cells were lysed in 0.5% Nonidet P-40 lysis buffer for immunoprecipitation of stimulated CD4 T cells and in PLC lysis buffer (50 mm Hepes-NaOH, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EDTA, protease inhibitor) for Western blot of whole cell lysate. Western blots of phosphotyrosine were performed as described in Ref. 29Asai K. Hachimura S. Kimura M. Toraya T. Yamashita M. Nakayama T. Kaminogawa S. J. Immunol. 2002; 169: 4723-4731Crossref PubMed Scopus (39) Google Scholar. Other Western blots were performed as follows. Immunoblots were blocked with 5% skim milk in TBS-Tween for 1 h, followed by probing with primary antibody for1hat room temperature or overnight at 4 °C. After washing with TBS-Tween three times, immunoblots were incubated with secondary antibody for 1 h. Immunoblots were then washed with TBS-Tween three times, and detections were performed with ECL (Amersham Biosciences). Early Apoptotic and Dead Cell Detection by Fluorescence-activated Cell Sorting—Splenocytes were enriched for CD4-positive cells with anti-CD4 microbeads and magnetic separation columns, according to the manufacturer's recommended protocol. Cells were then labeled with FITC-conjugated annexin V (Roche Applied Science), PI, and APC-conjugated anti-mouse CD4 mAb according to the manufacturer's instructions. Their staining profiles were analyzed by fluorescence-activated cell sorting LSR and CellQuest software (BD Biosciences, Mountain View, CA). DNA Fragmentation Assay—Triplicate 4 × 106 purified T cells were subjected to a DNA fragmentation assay with diphenylamine reagent as previously described in Ref. 34Kaji T. Kaieda I. Hisatsune T. Kaminogawa S. Nitric Oxide. 2002; 6: 125-134Crossref PubMed Scopus (25) Google Scholar. Orally Tolerant CD4 T Lymphocytes Are Induced by Long Term Ag Administration—To obtain orally tolerant splenic CD4 T cells, OVA323–339-specific TCR transgenic mice (OVA23-3) were fed with dietary Ag (20% egg white diet) for 4 weeks. As shown in our previous study (29Asai K. Hachimura S. Kimura M. Toraya T. Yamashita M. Nakayama T. Kaminogawa S. J. Immunol. 2002; 169: 4723-4731Crossref PubMed Scopus (39) Google Scholar), these CD4 T cells exhibited reduced proliferation and reduced IL-2, IL-4, and IFN-γ cytokine production when incubated with OVA and APCs in vitro (Fig. 1, A and B). These characteristics of CD4 T cells are evident after 4 consecutive weeks of oral administration of dietary Ag (data not shown). One known mechanism of tolerance induction is through the active suppression of regulatory T lymphocytes that subsequently produces negative regulatory cytokines such as transforming growth factor-β and IL-10; however, the production of IL-10 was not detected in this experimental system (Fig. 1C). These results indicated that the splenic CD4 T lymphocytes of OVA23-3 mice freely fed the egg white diet for 4 weeks demonstrated a form of oral tolerance that was not induced by active suppression. In Vivo Protein Expression Analysis of Orally Tolerant CD4 T Lymphocytes with 2-DE—To characterize protein expression levels of orally tolerant CD4 T cells, we compared these cells with unsensitized CD4 T cells obtained from control diet-fed OVA23-3 mice by 2-DE. We used whole cell lysates to represent the in vivo state of cellular proteins and to make an accurate comparative study. We also utilized narrow range immobilized pH gradient gels for the first dimension electrophoresis to obtain high resolution separation. Fig. 2A shows silver-stained two-dimensional separated gels from both control and orally tolerant CD4 T cells. The arrowheads show the up-regulated protein spots when the two gels were compared. We also used luminescent staining, which is known to deliver a wide quantitation range (35Berggren K. Chernokalskaya E. Steinberg T.H. Kemper C. Lopez M.F. Diwu Z. Haugland R.P. Patton W.F. Electrophoresis. 2000; 21: 2509-2521Crossref PubMed Scopus (329) Google Scholar), but could not produce the same sensitivity as observed using silver staining (data not shown). In relation to the detected spots, similar results were obtained when comparing spot intensities between gels. We detected 42 changed spots between pH 4.5 and 6.7, of which 26 were increased and 16 decreased for orally tolerant CD4 T cells. Of the differentially expressed spots, we identified 35 by peptide mass fingerprinting. Table I shows the peptide mass fingerprint results, which contain experimental and theoretical pI and molecular weights, NCBI protein accession number, the number of matching peptides, and the amino acid coverage of the obtained peptides. The -fold induction of the protein spots (orally tolerant versus control CD4 T cells) are also shown. There were some unidentified spots that had no matching proteins in the data base to date, although we could identify specific digested peptide peaks, or in some cases, the amount of protein expressed was too little to be precisely identified by this method.Table IUp-regulated and down-regulated proteins in orally tolerant CD4 T cellsSpot numberProtein spot-fold inductionIdentified proteinNCBI accession no.Theor. pITheor. MWEx. pIEx. MWPeptide matchesA.A. coveragekDa14.44Lipocortin 1 (Annexin I)1139456.638,6926.53983124.05Caspase-3 (CPP-32)24935276.531,4756.43273133.99EIA128348915.942,5755.740112743.41Annexin VI1139635.375,8875.370112853.39Unidentified5.42262.96Stathmin (Phosphoprotein p19)17115605.817,2755.41752672.79Unidentified6.52582.63Secretory protein precursor (YM-1)111408775.444,4595.445103292.56Caspase-1 (Interleukin-1β-converting enzyme)2663225.745,6415.7491125102.54RIKEN cDNA 1110001E24 gene180434465.146,8535.146929112.41Protein for MGC:37456193535935.236,0845.2451552122.38Carbonate dehydratase (EC 4.2.1.1)II197451816.529,0336.529737132.35ActinaActin is predicted caspase-cleaved fragment, described in (Ref. 43). Amino acid coverage means the coverage of predicted fragment.716195.341,7375.331527142.04Unidentified5.420151.89Δ-aminolevulinate acid dehydratase1228346.336,0246.136628161.79Unidentified5.865171.73Unidentified5.437181.62Similar to HSCARG protein209879906.434,3766.2321035191.62Myo-inositol I-phosphate synthase A196244986.060,9325.962725201.61Peroxiredoxin 430247156.731,0535.827840211.53Homolog to stomatin-like protein 2 (SLP-2)123827778.938,3855.939533221.52Similar to replication factor C (activator 1) 4130971236.339,8676.339725231.48ActinaActin is predicted caspase-cleaved fragment, described in (Ref. 43). Amino acid coverage means the coverage of predicted fragment.716195.341,7375.429742241.45PKCθ-interacting protein PICOT68409495.437,7835.440528251.40Heterogeneous nuclear ribonucleoprotein H22530415.949,2005.752926261.36Heterogeneous nuclear ribonucleoprotein K122305525.350,9945.365927270.75Similar to hypothetical protein HT036193542636.030,4496.129529280.75Unidentified6.536290.73PDZ and LIM domain 1 (elfin)134359396.435,7756.237933300.71Isovaleryl coenzyme A dehydrogenase128366558.346,2276.2421030310.71Nit protein 293671146.430,5026.130737320.70Calcium-binding protein p2224934735.022,4325.022427330.70Adenosine deaminase1133405.539,9925.541735340.68Lysophospholipase II45894536.724,7946.325531350.67RIKEN cDNA 2310041H06 gene128447148.923,7076.324425360.65Glutathione peroxidase 1128320906.116,4835.825526370.57STE20-like kinase MST-184898675.155,5425.1581534380.56GADS66854916.136,8106.1371030390.56Protein phosphatase 1, catalytic subunit, α isoform159287325.937,5405.938827400.56GADS66854916.136,8105.9371350410.48Unidentified6.254420.48Cofilin1096758.218,5606.118642a Actin is predicted caspase-cleaved fragment, described in (Ref. 43Mashima T. Naito M. Noguchi K. Miller D.K. Nicholson D.W. Tsuruo T. Oncogene. 1997; 14: 1007-1012Crossref PubMed Scopus (218) Google Scholar). Amino acid coverage means the coverage of predicted fragment. Open table in a new tab Caspase Activities in Orally Tolerant CD4 T Lymphocytes— Up-regulated proteins in orally tolerant CD4 T cells included both caspase-1 and caspase-3. The caspases are a family of cysteine aspartic acid proteases that play a central role in the regulation of apoptosis (36Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6218) Google Scholar). Activation of members of the caspase family requires their cleavage; for example, the pro form of caspase-3 (32 kDa) is cleaved into its active form (17 or 20 kDa) (37Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T. Yu V.L. et al.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3837) Google Scholar). Furthermore, the effector caspases are responsible for the cleavage of target proteins. In our results of 2-DE gels, some of the proteins identified with decreased expression levels in orally tolerant CD4 T cells have been reported to undergo caspase-dependent cleavage: i.e. STE20-like kinase MST-1, which promotes apoptosis after cleavage (38Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (328) Google Scholar, 39Ura S. Masuyama N. Graves J.D. Gotoh Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10148-10153Crossref PubMed Scopus (140) Google Scholar), and GADS (40Berry D.M. Benn S.J. Cheng A.M. McGlade C.J. Oncogene. 2001; 20: 1203-1211Crossref PubMed Scopus (24) Google Scholar, 41Yankee T.M. Draves K.E. Ewings M.K. Clark E.A. Graves J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6789-6793Crossref PubMed Scopus (20) Google Scholar). In addition, actin is cleaved into 40-, 31-, 29-, and 15-kDa polypeptides by caspase-1 and -3 (42Mashima T. Naito M. Fujita N. Noguchi K. Tsuruo T. Biochem. Biophys. Res. Commun. 1995; 217: 1185-1192Crossref PubMed Scopus (204) Google Scholar, 43Mashima T. Naito M. Noguchi K. Miller D.K. Nicholson D.W. Tsuruo T. Oncogene. 1997; 14: 1007-1012Crossref PubMed Scopus (218) Google Scholar), and we found that the 31- and 29-kDa cleaved forms of actin were increased in orally tolerant CD4 T cells (Table I). These findings also suggest that active caspase-3 or -1 was up-regulated in orally tolerant CD4 T cells, although we only detected the pro form of caspase-3 or -1 up-regulation in 2-DE gels. Therefore, we next analyzed caspase-3 cleavage by Western blotting. As expected, the pro form and active form of caspase-3 were increased in orally tolerant CD4 T cells (Fig. 3A). These results suggest that caspases play an important role in orally tolerant CD4 T cells. Although the activation of caspases contributes to apoptosis, there are some reports that caspase-3 activation occurs in nonapoptotic T cells (44Miossec C. Dutilleul V. Fassy F. Diu-Hercend A. J. Biol. Chem. 1997; 272: 13459-13462Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 45Wilhelm S. Wagner H. Hacker G. Eur. J. Immunol. 1998; 28: 891-900Crossref PubMed Scopus (103) Google Scholar, 46Alam A. Cohen L.Y. Aouad S. Sekaly R.P. J. Exp. Med. 1999; 190: 1879-1890Crossref PubMed Scopus (360) Google Scholar). We therefore examined whether orally tolerant CD4 T cells underwent apoptosis. As shown in Fig. 3B, very few apoptotic (annexin V-positive) and necrotic (PI-positive) cells were identified in both control
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