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• W2044332641 abstract "Intercellular adhesion molecule-3 (ICAM-3), a ligand for β2 integrins, elicits a variety of activation responses in lymphocytes. We describe a functional mapping study that focuses on the 37-residue cytoplasmic region of ICAM-3. Carboxyl-terminal truncations delineated portions involved in T cell antigen receptor costimulation, homotypic aggregation, and cellular spreading. Truncation of the membrane distal 25 residues resulted in loss of T cell antigen receptor costimulation as determined by interleukin 2 secretion. Aggregation and cell spreading were sensitive to truncation of the membrane distal and proximal thirds of the cytoplasmic portion. Phosphoamino acid analysis revealed that ICAM-3 from activated cells contained phosphoserine and phosphopeptide mapping identified Ser489 as a site of phosphorylation in vivo. Mutation of Ser489 or Ser515 to alanine blocked interleukin 2 secretion, aggregation and cell spreading, while mutation of other serine residues affected only a subset of functions. Ser489 was a phosphorylation site in vitro for recombinant protein kinase Cθ. Finally, treatment of Jurkat cells with chelerythrine chloride, a protein kinase C inhibitor, prevented ICAM-3-triggered spreading. This study delineates separable regions and amino acid residues within the cytoplasmic portion of ICAM-3 that are important for T cell function. Intercellular adhesion molecule-3 (ICAM-3), a ligand for β2 integrins, elicits a variety of activation responses in lymphocytes. We describe a functional mapping study that focuses on the 37-residue cytoplasmic region of ICAM-3. Carboxyl-terminal truncations delineated portions involved in T cell antigen receptor costimulation, homotypic aggregation, and cellular spreading. Truncation of the membrane distal 25 residues resulted in loss of T cell antigen receptor costimulation as determined by interleukin 2 secretion. Aggregation and cell spreading were sensitive to truncation of the membrane distal and proximal thirds of the cytoplasmic portion. Phosphoamino acid analysis revealed that ICAM-3 from activated cells contained phosphoserine and phosphopeptide mapping identified Ser489 as a site of phosphorylation in vivo. Mutation of Ser489 or Ser515 to alanine blocked interleukin 2 secretion, aggregation and cell spreading, while mutation of other serine residues affected only a subset of functions. Ser489 was a phosphorylation site in vitro for recombinant protein kinase Cθ. Finally, treatment of Jurkat cells with chelerythrine chloride, a protein kinase C inhibitor, prevented ICAM-3-triggered spreading. This study delineates separable regions and amino acid residues within the cytoplasmic portion of ICAM-3 that are important for T cell function. ICAM-3 1The abbreviations used are: ICAM, intercellular adhesion molecule; LFA-1, lymphocyte function antigen-1; IL2, interleukin 2; TCR, T cell receptor; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PKC, protein kinase C; APC, antigen-presenting cell; PCR, polymerase chain reaction; HA, hemagglutinin; MHC, major histocompatibility complex; PMSF, phenylmethylsulfonyl fluoride; PMA, phorbol 12-myristate 13-acetate. (CD50) is a member of the Ig superfamily sharing sequence and functional attributes with ICAM-1, -2, -4 (Landsteiner-Weiner blood group glycoprotein), and -5 (telencephalin). ICAM-3 binds to LFA-1 (CD11a/CD18) and the newly described integrin αd/CD18 (1Van der Vieren M. Le Trong H. Wood C.L. Moore P.F. St. John T. Staunton D.E. Gallatin W.M. Immunity. 1995; 3: 683-690Abstract Full Text PDF PubMed Scopus (232) Google Scholar). It is constitutively expressed at high levels by most hematopoietic cells, leading to the suggestion that it is involved in early activation steps of an inflammatory response (2Vilella R. Mila J. Lozano F. Alberola-Ila J. Places L. Vives J. Tissue Antigens. 1990; 36: 203-210Crossref PubMed Scopus (30) Google Scholar, 3de Fougerolles A.R. Springer T.A. J. Exp. Med. 1992; 175: 185-190Crossref PubMed Scopus (398) Google Scholar, 4Vazeux R. Hoffman P.A. Tomita J.K. Dickinson E.S. Jasman R.L. St. John T. Gallatin W.M. Nature. 1992; 360: 485-488Crossref PubMed Scopus (183) Google Scholar). ICAM-3 has been functionally characterized with respect to the five Ig-like extracellular domains. The first amino-terminal domain binds LFA-1 via conserved residues also found in ICAM-1 (5Sadhu C. Lipsky B. Erickson H.P. Hayflick J. Dick K.O. Gallatin W.M. Staunton D.E. Cell Adhes. Commun. 1994; 2: 429-440Crossref PubMed Scopus (39) Google Scholar). These conserved sequences of ICAM-3 and -1 may bind to distinct sites of the I domain of LFA-1, suggesting that non-conserved domain 1 residues might contribute to integrin binding (6Binnerts M.E. van Kooyk Y. Edwards C.P. Champe M. Presta L. Bodary S.C. Figdor C.G. Berman P.W. J. Biol. Chem. 1996; 271: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 7van Kooyk Y. Binnerts M.E. Edwards C.P. Champe M. Berman P.W. Figdor C.G. Bodary S.C. J. Exp. Med. 1996; 183: 1247-1252Crossref PubMed Scopus (41) Google Scholar). Numerous intracellular signaling events have also been observed to be affected by ICAM-3 engagement. Specifically, activation of intracellular calcium flux and stimulation of tyrosine kinase activity possibly via non-receptor tyrosine kinases p56 lck and p59 fyn were seen (8Juan M. Vinas O. Pino-Otin M.R. Places L. Martinez-Caceres E. Barcelo J.J. Miralles A. Vilella R. de la Fuente M.A. Vives J. J. Exp. Med. 1994; 179: 1747-1756Crossref PubMed Scopus (52) Google Scholar, 9Arroyo A.G. Campanero M.R. Sanchez-Mateos P. Zapata J.M. Ursa M.A. del Pozo M.A. Sanchez-Madrid F. J. Cell Biol. 1994; 126: 1277-1286Crossref PubMed Scopus (88) Google Scholar). ICAM-3 engagement has also been observed to up-regulate β1 and β2 integrin function, and to trigger phosphorylation of the cyclin-dependent kinase cdc2 (10Campanero M.R. del Pozo M.A. Arroyo A.G. Sanchez-Mateos P. Hernandez-Caselles T. Craig A. Pulido R. Sanchez-Madrid F. J. Cell Biol. 1993; 123: 1007-1016Crossref PubMed Scopus (135) Google Scholar, 11Campanero M.R. Sanchez-Mateos P. del Pozo M.A. Sanchez-Madrid F. J. Cell Biol. 1994; 127: 867-878Crossref PubMed Scopus (73) Google Scholar, 12Chirathaworn C. Tibbetts S.A. Chan M.A. Benedict S.H. J. Immunol. 1995; 155: 5479-5482PubMed Google Scholar). Little information, however, is available regarding the molecular mechanisms of these phenomena. Here we report that ICAM-3 engagement initiates several distinct aspects of lymphocyte function, which involve the 37-amino acid cytoplasmic portion. For these analyses, we developed and characterized an ICAM-3-deficient human T-leukemic Jurkat cell line. Using these cells and gene transfer techniques, a functional map of the cytoplasmic region of ICAM-3 with respect to TCR accessory molecule function, homotypic aggregation, and cell spreading was generated. These data pinpoint serine residues, particularly serine 489, as critical for ICAM-3 function. Jurkat 77 (J77, a gift from Dr. S. Burakoff, Dana Farber Cancer Research Institute, Boston, MA) and the ICAM-3-deficient J77.50.3 cells were maintained in RPMI complete medium (RPMI supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine, and 1 mm sodium pyruvate) in humidified 5% CO2 at 37 °C. Murine mAb used in this study were as follows. Anti-ICAM-3 (CD50) mAb ICR1.1 (IgG2a), ICR2.1 (IgG1), ICR9.2 (IgG2a), and anti-ICAM-1 (CD54) mAb 18E3D (IgG1) were generated by the ICOS hybridoma facility. Hybridoma lines secreting anti-TCR (CD3e) OKT3 (IgG2a), anti-CD11a TS1/22 (IgG1), anti-CD18 TS1.18 (IgG1), anti-CD45 4B2 (IgG2a), and anti-MHC class I W6/32 (IgG2a) were obtained from American Type Culture Collection, Rockville, MD. These mAb were purified by protein A column chromatography of mouse ascites fluid. Purified mAb were dialyzed against and stored in PBS. Isotype-matched control mAb used were UPC10 (IgG1) and MOPC 21 (IgG2a) (Sigma). Anti-HA mAb 12CA5 (IgG2a) was from Boehringer Mannheim. Fluorescein isothiocyanate-conjugated sheep anti-mouse IgG F(ab′)2 was purchased from Sigma. A variant of Jurkat 77 cells deficient in the expression of ICAM-3 (J77.50.3) was generated by two rounds of indirect staining and cell sorting using a mixture of ICR1.1 and 9.2 mAb (4Vazeux R. Hoffman P.A. Tomita J.K. Dickinson E.S. Jasman R.L. St. John T. Gallatin W.M. Nature. 1992; 360: 485-488Crossref PubMed Scopus (183) Google Scholar). J77.50.3 cells were compared with the parental line for surface expression of numerous membrane proteins by indirect cytofluorometry (FACSCAN, Becton-Dickinson, Mountain View, CA) and found to exhibit similar levels for all except ICAM-3. Ten micrograms of total RNA isolated from parental J77 and J77.50.3 cells was subjected to blotting, hybridization, and washing as described (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Labeled probes were generated by random priming of the entire ICAM-3 or glyceraldehyde-3-phosphate dehydrogenase cDNA (14Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). Coding sequences for HA epitope-tagged ICAM-3 proteins were generated as described (15Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar). To engineer epitope-tagged full-length ICAM-3 construct, three separate PCR fragments that encoded 1) the signal sequence (preceded by a unique HindIII site and Kozak sequence), 2) Ig domains (IgD) I-II of ICAM-3, and 3) a triple (3×) influenza hemagglutinin (HA) epitope tag sequence were synthesized and gel-purified (16Field J. Nikawa J. Broek D. MacDonald B. Rodgers L. Wilson I.A. Lerner R.A. Wigler M. Mol. Cell. Biol. 1988; 8: 2159-2165Crossref PubMed Scopus (733) Google Scholar). The fragments were combined using PCR in the following order: ICAM-3 signal sequence, HA tag, and ICAM-3 IgD I-II. This product was ligated as a HindIII/ScaI fragment with a ScaI/EcoRI cDNA fragment containing the remainder of the ICAM-3 coding sequence into theHindIII/EcoRI sites of expression vector pMH-neo and all PCR products sequenced (17Hahn W.C. Menzin E. Saito T. Germain R.N. Bierer B.E. Gene ( Amst. ). 1993; 127: 267-268Crossref PubMed Scopus (35) Google Scholar). Cytoplasmic region deletions were generated as follows. The region of coding sequence for the extracellular domains described above contained on a HindIII/SacI fragment was combined withSacI/EcoRI PCR fragments encoding cytoplasmic domain truncations and ligated to theHindIII/EcoRI sites of pMH-neo. The PCR fragments were synthesized using the following primers: 1) 5′ common anchoring primer CATAATGGTACTTATCAGTGC, and 2) 3′ primers D505 (−1/3CT), ATATAGCGGCCGCGGATCCTCACTGCATAGACGTGAG; D493 (−2/3CT), ATATAGCGGCCGCGGATCCTCACCTAACATGGTAACT; and D484 (−CT), ATCACTATGCGGCCGCTCAGTGTCTCCTGAAGACGTACAT. Primer D484 contained a change at codon 483 to increase the membrane anchor region of the maximal cytoplasmic region truncation. Amino acid numbering uses the mature amino terminus for the first residue. To generate point mutations, the ICAM-3 cDNA was subcloned as aNotI/EcoRI fragment into M13 BM21 replicative form DNA (Boehringer Mannheim) and primers used for mutagenesis by the Kunkel method were designed to make alanine changes at the following codons: serine 487, serine 489, leucine 499, serine 496, serine 503, and serine 515 (18Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). Leucine 499 was chosen as a control for mutational effect, since it is not a potential phosphorylation site and a conservative change to alanine is expected to maintain similar overall charge. All mutants were sequenced, and each was subcloned as aSacI/EcoRI fragment along with theHindIII/SacI fragment described above into pMH-neo. J77.50.3 cells (107) in 0.2 ml of PBS were electroporated in the presence of 100 μg/ml plasmid DNA using a BTX 6000 device (126 V, 1710 microfarads, 72 milliamps, 0.2-cm electrode gap). Drug-resistant cells (G418, 1.25 mg/ml) were screened by indirect fluorescence flow cytometry with ICR1.1, OKT3, or isotype-matched control antibody. Two independent cell lines were chosen for functional studies from each transfection based on similar mean channel fluorescence measurements. To enrich for cell lines that expressed higher mean ICAM-3 levels, the drug-resistant lines were panned on mAb-coated plastic. Bacteriologic Petri plates were incubated with 8 ml of ICR2.1 (10 μg/ml) in PBS for 2 h at 37 °C. The plates were rinsed with PBS and cells seeded onto the mAb-coated plastic surface for 8 min at 25 °C. To remove non-adherent cells, the plates were rocked and aspirated. The plates were rinsed with PBS, checked visually to determine the absence of non-adherent cells, and adherent cells removed by trituration. Cells harvested in this manner were expanded and subjected to indirect fluorescence analysis using flow cytometry. Plates (96-well, Corning 25860) were coated with OKT3 (0.5 μg/ml, 50 μl/well) in PBS for 16 h at 4 °C. The coating was removed and replaced with PBS alone or mAb at 10 μg/ml in PBS and incubated at 37 °C for 2 h. Wells were rinsed twice with PBS and 2 × 105 cells added in 0.25 ml of RPMI/well. Plates were incubated at 37 °C for 16 h. Conditioned medium from duplicate wells was pooled, diluted serially, and assayed for IL2 concentration by enzyme-linked immunosorbent assay (Biosource International, Camarillo, CA). A dose for OKT3 (25 ng/well) and ICR1.1 (500 ng/well) was chosen for co-stimulation experiments. Assays were repeated a minimum of three times, with similar results observed in each experiment. Dishes (ΔT, 0.5-mm glass; Bioptechs Inc., Butler, PA) were coated with 0.5 ml of mAb in PBS (10 μg/ml). Dishes was incubated at 37 °C for 2 h and rinsed twice with PBS. Cells (2 × 104) were seeded onto coated surfaces for 15 min at 37 °C. Plates were held at 37 °C in the Bioptechs stage insert while being photographed with Ilford Pan F film using a Nikon Diaphot microscope and DIC optics. For PKC inhibitor studies, chelerythrine chloride (in Me2SO) was added to cells at a final concentration of 50 μm and incubated at 37 °C for 10 min prior to seeding into coated dishes. Cells were pelleted and resuspended at 8 × 105/ml in complete medium and 0.25 ml distributed to duplicate wells of a 96-well flat bottom plate. mAb ICR1.1 or MOPC 21 control were added to a final concentration of 10 μg/ml. After 1 h of incubation at 37 °C, the percentage of aggregated cells was determined as described previously (19Lorenz H.M. Harrer T. Lagoo A.S. Baur A. Eger G. Kalden J.R. Cell. Immunol. 1993; 147: 110-128Crossref PubMed Scopus (37) Google Scholar). Quantitative determinations were made by counting free cells in five separate squares of a gridded ocular centered over the well at 125 × magnification. Percent aggregation = {1 − (no. of free cells experimentally treated/no. of free cells control-treated)} × 100. Cells (5 × 107) were starved of methionine and cysteine for 1 h prior to labeling with 100 μCi/ml [35S]methionine and -cysteine (Tran35S-Label; ICN Biochemicals, Irvine, CA) in RPMI, 10% dialyzed fetal bovine serum. Cells for phosphorylation studies were rinsed in phosphate-free RPMI and labeled with 0.5 mCi of inorganic 32P for 4 h at 37 °C. Labeled cell pellets were suspended in 1 ml of cold lysis buffer (PBS containing 1% Triton X-100, 1 mm PMSF, 1 mm Na3VO4, 1 mmNa2MoO4) and incubated on ice for 20 min with occasional rocking. The insoluble fraction was pelleted by centrifugation in a table top microcentrifuge. Soluble proteins were transferred to a fresh tube and 0.1 ml of Sepharose 4CL beads added (50% slurry equilibrated in lysis buffer without PMSF; Pharmacia Biotech Inc., Uppsula). The tube was rocked for 16 h, after which the beads were briefly spun down. The clarified supernatant was transferred to a fresh tube and antibody added to 10 μg/ml final concentration. Immune complexes were formed by incubation on ice for 1 h and harvested by incubation with protein A beads. The immune complexes were pelleted and washed two times with 1 ml of cold 1% Triton X-100, 1 m NaCl, 1 mm PMSF, 1 mm Na3VO4, 1 mmNa2MoO4, and once with 1 ml of cold lysis buffer. The remaining proteins were eluted by addition of reducing SDS-PAGE loading buffer, boiled for 5 min, and separated by gel electrophoresis. Immune complexes of labeled proteins were separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Autoradiography of gels containing32P-labeled proteins was conducted using X-Omat film (Eastman Kodak Corp.) with a single intensifying screen at −70 °C. Gels of 35S-labeled proteins were impregnated with fluor and exposed to film at −70 °C. Autoradiographs were used to localize32P-labeled ICAM-3. Bands were excised from the polyvinylidene difluoride sheet and the proteins partially acid hydrolyzed and separated as described in (20Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Briefly, the samples were dried in vacuo and resuspended in 6 μl of pH 1.9 buffer containing unlabeled phosphoamino acid standards (Sigma). A portion of each sample, representing equal Cerenkov counts, was spotted on cellulose TLC plates and phosphoamino acids separated by high voltage thin layer electrophoresis (HTLE-7000; CBS Scientific, Del Mar, CA). After ninhydrin staining of the standards, the plates were exposed to film for autoradiography. Phosphorylation sites were determined by tryptic peptide mapping usingin vivo 32P-labeled proteins. Labeled protein bands were incubated with l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington Biochemical Corp., Freehold, NJ) for 18 h. The resulting peptide mixture was spotted on a TLC plate and subjected to charge separation and ascending chromatography. Human protein kinase Cθ was amplified by PCR with a primer that contained a 6-histidine tag at the carboxyl terminus of the protein coding sequence as described (21Baier G. Baier-Bitterlich G. Meller N. Coggeshall K.M. Giampa L. Telford D. Isakov N. Altman A. Eur. J. Biochem. 1994; 225: 195-203Crossref PubMed Scopus (78) Google Scholar). The cDNA was subcloned into a BACMID vector (Life Technologies Inc.), and recombinant virus was generated as described by the manufacturer. Infected Sf9 cells were lysed in hypotonic buffer (20 mmTris, pH 7.5, 250 mm sucrose, 5 mm EDTA, 5 mm EGTA, 100 μg/ml each aprotinin and leupeptin, 1 mm aminoethyl benzenesulfonyl floride) by Dounce homogenization. Active soluble protein kinase was separated from insoluble material by centrifugation (16,000 × g) and 0.04-ml assays performed using 50 μg/ml total protein and 100 μm substrate peptide as described (21Baier G. Baier-Bitterlich G. Meller N. Coggeshall K.M. Giampa L. Telford D. Isakov N. Altman A. Eur. J. Biochem. 1994; 225: 195-203Crossref PubMed Scopus (78) Google Scholar). The following substrates were synthesized: ICAM-3 CT, amino acids 482–518 (REHQRSGSYHVREESTYLPLTSMQPTEAMGEEPSRAE), SCR CT (ARSTEQQGMYAESESEELRPGYPEHRSTHTMLPRSVE), SGS (biotin-FREHQRSGSYHVREE), and SGS-P (biotin-FREHQRSGS(PO4)YHVREE). Jurkat cells (J77) examined by indirect fluorescence cytometry with antibodies to ICAM-3 routinely display two populations of cells (Fig. 1 A). The bulk (97%) of these are ICAM-3-positive, while a small population (3%) is ICAM-3-negative. Enrichment of the ICAM-3-negative population by sequential rounds of cell sorting generated a population displaying >97% ICAM-3-negative cells that we have termed J77.50.3. RNA analysis showed that, in J77.50.3 cells, synthesis of the 2.2-kb ICAM-3 message was below detectable levels (Fig. 1 B). Surface expression of numerous proteins was assessed in J77.50.3 and the parental J77 line. Both populations exhibited similar fluorescence profiles for all mAb studied including CD3e, CD11a, CD18, and CD45 (Fig. 1 A), indicating that J77.50.3 cells were similar to J77 except for the ICAM-3 deficiency. Optimal T cell activation is thought to require two signals: one from the antigen receptor and the other from one or more of a large number of accessory molecules including ICAM-3 (22Liu Y. Linsley P.S. Curr. Opin. Immunol. 1992; 4: 265-270Crossref PubMed Scopus (230) Google Scholar). To confirm that ICAM-3/TCR engagement was costimulatory, Jurkat T cells were seeded onto ICR1.1 coimmobilized with increasing concentrations of OKT3. A dose-dependent increase in IL2 production was observed (Fig. 2 A). Cells exposed to either immobilized mAb alone showed no induction of IL2 secretion. To determined if loss of ICAM-3 expression would impair costimulation, J77.50.3 cells were seeded into mAb-coated wells under conditions that stimulated the parental cells. ICAM-3-deficient J77.50.3 did not secrete IL2 (Fig. 2 B). Both cell lines responded to a greater concentration of OKT3 by secreting IL2. Neither cell line responded to a combination of anti-ICAM-1 mAb (18E3D) and OKT3. These data reveal that TCR signaling and the synthetic machinery for IL2 production in J77.50.3 cells was intact. Further, co-engagement of ICAM-1 and CD3 was insufficient to produce IL2. To evaluate whether ICAM-3 expression in J77.50.3 cells would complement the phenotypic defect, cells were transfected with either a control vector or HA-tagged ICAM-3 (ICAM-3FL). Cells were selected, and several independent lines that maintained stable surface expression were identified (Fig. 3 A). Inclusion of the HA tag allowed for validation that the expressed form of ICAM-3 in the transfected cells was from the introduced DNA construct rather than re-expression of the endogenous gene. Indeed, surface staining for either ICAM-3 or HA tag epitopes showed similar levels of fluorescence in the populations (Fig. 3 A). While the control-transfected lines lacked the ability to be stimulated by the co-immobilized mAb, J77.50.3 cells expressing ICAM-3FL responded to the costimuli by secreting IL2 into the medium as did the parental J77 cells (Fig. 3 B). Therefore, expression of ICAM-3 by J77.50.3 cells restored their ability to respond to ICAM-3/TCR costimulation. J77.50.3 cells expressing the following HA epitope-tagged cytoplasmic tail truncations were generated to grossly map the cytoplasmic region of ICAM-3: −1/3CT (Gln505terminus), −2/3CT (Arg493 terminus), and −CT (His484 terminus) (Fig. 4). Characterization of cells expressing each deletion included monitoring surface expression (Fig. 5 Aand Table I) and immunoprecipitation from lysates of cells that had been metabolically labeled with35S (Fig. 5 B). SDS-PAGE analysis showed that the relative migration of the truncated proteins was of the expected sizes of ∼120–140 kDa.Figure 5Surface cytofluorometric and SDS-PAGE analysis of ICAM-3 cytoplasmic tail truncations expressed in J77.50.3 cells. A, ICR1.1 (filled histogram trace) or IgG2a control (outlined histogram trace) staining of cells expressing −1/3CT (left), −2/3CT (center), or −CT (right). B, analysis of ICAM-3FL and truncations from 35S metabolically labeled J77.50.3 cell transfectants by immunoprecipitation and SDS-PAGE. Cell extracts from vector control, ICAM-3FL, −1/3CT, −2/3CT, and −CT (lanes 1–5, respectively) were analyzed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table ICharacterization of different J77.50.3 transfectantsCell lineMean fluorescence intensity1-aMean fluorescence intensity for transfectants was determined from live-gated cell populations and expressed in arbitrary units.IL2 secretion (fold stimulation)1-bIL2 secreted from cells seeded onto coimmobilized mAb after a 16-h incubation was measured by enzyme-linked immunosorbent assay. Wells were treated with 500 ng/well (ICR1.1 or 18E3D) and 25 ng/well OKT3. Fold stimulation = (IL2 secreted from cells on coimmobilized ICR1.1/OKT3)/(IL2 secreted from cells on coimmobilized 18E3D/OKT3). Results are the means of repeated experiments (n = 3–6) with two cell lines for each truncation/mutation.Percent aggregation1-cPercent aggregation is based on extrapolation by counting free cells remaining after a 60-min incubation at 37 °C in medium containing ICR1.1 or MOPC21 isotype control (n = 3–5).Percent spread cells1-dCell spreading on ICR1.1-coated surfaces is expressed as percent of input cells. After incubation and fixation, 300 cells were counted for each represented cell line. Cells considered spread showed thinned, darkened cytoplasm. Cells considered not spread were refractile and bright (n = 2–5).Vector120.5216 ± 40FL1565.458 ± 387 ± 10−1/3CT1664.829 ± 21-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.26 ± 11-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.−2/3CT1322.229 ± 11-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.33 ± 31-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.−CT2222.215 ± 21-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.16 ± 11-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.Ser487 → Ala1742.061 ± 977 ± 5Ser489 → Ala1532.912 ± 21-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.10 ± 11-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.Ser496 → Ala1576.058 ± 733 ± 31-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.Leu499 → Ala1586.057 ± 474 ± 8Ser503 → Ala1371.066 ± 1536 ± 21-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.Ser515 → Ala2622.115 ± 31-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.14 ± 31-eThese values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05.1-a Mean fluorescence intensity for transfectants was determined from live-gated cell populations and expressed in arbitrary units.1-b IL2 secreted from cells seeded onto coimmobilized mAb after a 16-h incubation was measured by enzyme-linked immunosorbent assay. Wells were treated with 500 ng/well (ICR1.1 or 18E3D) and 25 ng/well OKT3. Fold stimulation = (IL2 secreted from cells on coimmobilized ICR1.1/OKT3)/(IL2 secreted from cells on coimmobilized 18E3D/OKT3). Results are the means of repeated experiments (n = 3–6) with two cell lines for each truncation/mutation.1-c Percent aggregation is based on extrapolation by counting free cells remaining after a 60-min incubation at 37 °C in medium containing ICR1.1 or MOPC21 isotype control (n = 3–5).1-d Cell spreading on ICR1.1-coated surfaces is expressed as percent of input cells. After incubation and fixation, 300 cells were counted for each represented cell line. Cells considered spread showed thinned, darkened cytoplasm. Cells considered not spread were refractile and bright (n = 2–5).1-e These values were compared with the values from the full-length ICAM-3-expressing cells using one-way analysis of variance (ANOVA) and found to be significantly different. Values forp = <0.05. Open table in a new tab The truncations were tested for their ability to trigger J77.50.3 cells to secrete IL2 when costimulated with anti-ICAM-3/TCR mAb. Conditioned media from cells expressing either ICAM-3FL or −1/3CT forms showed 5.4- and 4.8-fold induction, respectively, when normalized for IL2 secretion in negative control-treated wells (Table I). Cells expressing either −2/3CT or −CT forms secreted about 60% less IL2 (2.2-fold each). All of the cell lines tested responded to a more concentrated dose of OKT3 alone by secreting similar levels of IL2 (data not shown). Immunoregulation of leukocytes has been hypothesized to occur via aggregate formation in which paracrine effects of cytokines (both positive and negative) regulate progression of a cellular immune response (23Mitchison A. Immunologist. 1995; 3: 259Google Scholar). J77.50.3 cells expressing ICAM-3FL treated with ICR1.1 mAb responded by forming aggregates (58% aggregated, Table I). This is not due to direct cross-linking of cells by mAb, since Fab fragments of ICR1.1 trigger ag" @default.
• W2044332641 created "2016-06-24" @default.
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• W2044332641 date "1997-08-01" @default.
• W2044332641 modified "2023-09-27" @default.
• W2044332641 title "Functional Mapping of the Cytoplasmic Region of Intercellular Adhesion Molecule-3 Reveals Important Roles for Serine Residues" @default.
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