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Physiologic Signaling in Normal Human T-cells: mRNA Phenotyping by Northern Blot Analysis and Reverse TranscriptionPolymerase Chain Reaction’ KENNETHJ. WIEDER,*GERDWALZ,*BERND ZANKER,* PRABODHSEHAJPAL, VIJAY K. SHARMA,EDWARDSKOLNIK,TERRY B. STROM,* AND MANIKKAM~UTHANTHIRAN *Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215 and The Rogosin Institute, Department ofMedicine and Biochemistry, Cornell University Medical College, New York, New York 10021 Received November IO. 1989; accepted January 16, 1990
Synergy between ionomycin and sn- l,2-dioctanoylglycerol (diCs) was shown at the level of lymphokine gene transcription. Transcriptional activation of interleukin-2 (IL-2), interferon-r (EN-r), and the protooncogene H-ras was accomplished by signaling highly purified normal human resting T-lymphocytes (T-cells) with diCs, a physiologic regulator of protein kinase C, and the calcium ionophore, ionomycin. Northern blot analysis of mRNA for early T-cell activation genesdemonstrated the synergism between diCs and ionomycin at the gene induction level. To amplify very low levels of IL-2 mRNA, sequential reverse transcription and polymerase chain reaction (RT-PCR) of T cell mRNA were used to demonstrate the capacity ofthe calcium signal (ionomycin) to promote low-level IL-2 transcription in normal human T-cells without additional signals. Cyclosporine (CsA) prevented diCs and ionomycin-induced expression of IL2, IFN-7, and H-ras genes. The completeness of its inhibitory effect was evident by the absence of IL-2 transcripts in CsA-treated cultures screened by the RT-PCR technique. CsA also prevented IL-2 and IF%-, gene expression in accessory cell-depleted T-cells stimulated by crosslinking the CD2 and CD3 antigens on the cell surface. Our observations demonstrate that a physiologic regulator of PKC, diCs, and cell surface crosslinking of the CD2 and CD3 antigen, promote gene expression in normal human quiescent T-cells independently of accessorycells, and that CsA prevents gene expression via a direct effect on T-cells. o 1990 Academic PXSS, IX.
INTRODUCTION An increase in the concentration of intracellular free calcium and the activation of protein kinase C (PKC) are thought to function as synergistic intracellular signals for the induced expression of growth-promoting genesin T-cells (1,2). However, much of the data that support this unifying theme for transcriptional activation have come largely from studies where preactivated T-cells (e.g., T-cell clones, T-leukemic lines etc.) rather than normal T-cells and nonphysiologic rather than physiologic activators of PKC have been utilized (3-5). Because the signaling requirements for normal, ’ This work was supported by Grants ROl-CA43977 and ROl-DK32929 (K.J.W., G.W., B.Z., and T.B.S.) from the National Institute of Health. 41 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
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primary T-cell activation may differ from leukemic or preactivated T-cells (6), including the documented dissimilarities between the nonphysiologic regulators of PKC such as phorbol estersand the physiologic diacylglycerols ( 1,7,8), additional studies were undertaken. In this report we sought to explore basic mechanisms concerning T-cell activation using the physiological activators of T-cells, ionomycin and diCs, and CD2/CD3 crosslinking, at the level of lymphokine and protooncogene transcription. Stimulation of highly purified T-cells with ionomycin, an accessorycell-dependent event, is augmented by physiologic activators of PKC (9). Since ionomycin and PKC activators are both required to stimulate T-cell activation, we wondered whether ionomycin activates one set of T-cell activation genesand another complementary set is activated by PKC activators or whether both signals are required to activate these genes. Similar information was sought in a system that crosslinked CD2-CD3 proteins with monoclonal antibodies (mAbs). Gene transcription data gathered by Northern blot analysis was supplemented by use of the polymerasechain reaction which allowed us to detect low abundance IL-2 transcription which was not obvious by Northern blot analysis. Here we report that synthetic and membrane permeant sn- 1,2-dioctanoylglycerol (diC*) transcriptionally activates interleukin-2 (IL-2) interferon-y (IFN-7) and H-ras genesin T-cells in the presence of ionomycin, independent of additional antigenic or accessorycell signals. Our results also show that cyclosporine (CsA), a clinically useful drug, prevents gene expression induced with the synergistic combination of diCs and ionomycin by a direct effect on T-cells. This new observation is extended by our additional demonstration that CsA prevents gene expression elicited by signaling of T-cells via the CD2 and CD3 antigens-an antigen-dependent T-cell activation pathway where the calcium ions and PKC activation contribute to an intracytoplasmic signal transduction pathway. MATERIALS AND METHODS T-Cell Isolation Human peripheral blood mononuclear cells (PBMCs) were isolated by FicollPaque density gradient centrifugation of venous blood obtained from healthy adult volunteers. T-cells were isolated from the PBMCs with a slight modification of a sequential four-step procedure which includes: (i) depletion of adherent cells by Sephadex G- 10 gel filtration, (ii) depletion of residual monocytes by treatment with 5 mM leucine methyl ester treatment, (iii) depletion of HLA-DR bearing cells and natural killer cells by treatment with anti-HLA-DR mAbs directed at the monomorphic component of HLA-DR antigen, 28-34 kDa), anti-NKH-1 A mAb (defining a 200-kDa molecule expressed on all NK cells but not abundantly expressedby other lymphoid or myeloid cells), and prescreened rabbit complement, and (iv) utilization of the SRBC-rosetting technique as the final preparative step for the isolation of T-cells. Fluorescence-activated cell sorter phenotypic analyses of a representative T-cell preparation: 99% CD2+, 98% CD3+, 0.36% NKH-I+, 0.34% HLA-DR+, 0.07% Leu-M3 (CD14+), 0.05% Leu-12 (CD19+), and 0.56% IL-2R (CD25+) cells. Activation with diC, and Ionomycin T-cells were ( lo6 cells/ml) suspended in RPM1 1640 medium supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin, 25 mM Hepes buffer, and 5% heat-
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inactivated fetal bovine serum (complete medium), and were incubated with predetermined optimum concentrations of diCs (10.0 pg/ml) and ionomycin (1 .O mM). The proliferative response is dependent upon signaling T-cells with both ionomycin and DAG, and is independent of additional antigenic or accessorysignals (9). Ionomycin (Streptomyces conglobatus [ATCC 3 1005]) was purchased from Calbiochem Biochemicals (San Diego, CA). DiCs was purchased from Sigma Chemical Co. (St. Louis, MO). Ionomycin and diC8 were initially dissolved in ethyl alcohol, and phosphate-buffered saline (PBS, Sigma) was then used to dilute the stock solution to the appropriate concentrations of each agent. T-cell Activation with Crosslinked Anti-CD2 and Anti-CD3 mAbs T-cells ( 1O6cells/ml) suspended in complete medium were incubated with 0.5 pug/ ml of anti-CD3 mAb antibodies (OKT3, Ortho Diagnostics Systems, Inc., Raritan, NJ), 0.5 lug/ml of anti-CD2 mAb (OKTll, Ortho Diagnostics) and 5.0 pg/ml of affinity-purified goat antibodies specific for mouse IgG (Southern Biotechnology Associates, Birmingham, AL). Polymerase Chain Reaction (PCR) PCR was employed to detect low abundance mRNA encoding IL-2 or actin in stimulated T cells. A 32-mer (“antisense”) oligonucleotide (5’-GGCAGAAGCTTGGCCTGATATGTTTTAAGTGGG-3’), 22 of which are complementary to the 3’ noncoding region of the IL-2 cDNA, and a second 26-mer (“sense”) oligonucleotide (5’-CAGTGTCTAGAAGAAGAACTCAAACC-3’) homologous to the IL-2 coding region at the XbaI restriction site were synthesized on an Applied Biosystems 38 1A DNA synthesizer. Actin oligomers were also synthesized which would amplify a 349basepair fragment of the actin transcript as described below. First-strand cDNA copies of the IL-2 and actin mRNAs were synthesized in a I O-p1reaction volume containing 10-pg of total RNA, 0.5 PM antisense oligonucleotide, and 200 units of M-MLV reverse transcriptase (Bethesda Research Laboratories, Bethesda, MD). The entire cDNA synthesis reaction volume was then combined in a 50-~1final reaction volume for PCR amplification containing 0.25 PMeach oligonucleotide primer, and I .5 units of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) under conditions suggested by the manufacturer. Forty cycles of PCR amplification were performed using a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). Each cycle consisted of 45 set for denaturation at 94°C 30 set for annealing at 55°C and 30 set for enzymatic primer extension at 72°C. Specificity of the amplified IL-2 and actin cDNAs fragments were determined by Southern blot analysis utilizing the IL-2 full-length cDNA, or identification of the appropriately sized ethidium bromide stained DNA fragment. In some experiments, first-strand cDNA copies were synthesized using oligo (dT) primers and M-MLV reverse transcriptase, and were then amplified with a sequencespecific primer pair using Taq polymerase. The PCR-generated products were then analyzed by agarosegel electrophoresis for the predicted size of cDNA fragment. RNA Isolation and Northern Blot Analysis Total RNA from T-cell preparations was isolated by the guanidinium isothiocyanate/cesium chloride method (10). Twenty micrograms of total RNA was run on a
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2.2 M formaldehyde/l% agarose gel (11). The gel was blotted directly onto Amersham’s Hybond nylon in 20X SSC(3.0 M NaC1/0.3 M Na citrate, pH 7). RNA transferred onto the filter was covalently immobilized to the support by exposure to 302 nm UV light for 4 min. The filter was prehybridized for 5 hr, then hybridized at 42°C to cDNA probes in a solution containing 50 mM sodium phosphate, pH 6.5, 50% formamide, 0.1% SDS, 8% dextran sulfate, 100 pg/ml denatured salmon sperm DNA, 3X Denhardt’s solution, 5X SSC. Following hybridization for 24 hr filters were washed twice in 0.2X SSC/O.1%SDS at room temperature for 10 min each, and twice at 55°C using the same solution for 20 min each. Filters are placed against Agfa RPI film for l-3 days. DNA Probes and Labeling IL-2 cDNA was provided by N. Farber (Biogen, Inc., Cambridge, MA) H-ras cDNA was obtained from ATCC, IL-2 receptor (Tat) cDNA was provided by Dr. Warren Leonard (National Institute of Health, Bethesda, MD), and interferon-y cDNA was provided by Dr. Steven Clark (Genetics Institute, Cambridge, MA). Complementary DNA was isolated from their plasmid vectors by restriction enzyme digestion and electroelution from agarosegel slices. The cDNAs were labeled with [32P]dCTP using the random priming procedure (12). Labeled cDNAs were added to hybridization solution at a concentration of 1 X lo6 cpm/ml of solution. RESULTS AND DISCUSSION Synergistic Transmembrane Signaling of T-Cells with die, and Ionomycin Diacylglycerols possessing eight carbon atom acyl chains are potent stimulators of PKC with the 3 hydroxyl group being essential for the successful interaction of diacylglycerol with PKC ( 13, 14). We therefore utilized diCs, a physiologic analogue of DAG, to activate PKC in highly purified normal human T-cells. Signaling of Tcells with diCs was accomplished in the presence of ionomycin, a highly specific calcium ionophore, because of the demonstrated synergy between the calcium signal and PKC activation for the elicitation of T-cell proliferation (9). The basis for this synergy has recently been explored in a thorough study of the signal transducing mechanisms that are activated by ionomycin or PMA, or both, in highly purified Tcells ( 15). Nonetheless this system has not been thoroughly explored at the transcriptional level. Moreover, we have utilized diCs rather than PMA because it is a physiological analog of diacylglycerol which also can activate PKC, without additional effectsexerted by phorbol esters(6, 14). Northern blot analysesdemonstrating the synergism between the physiological regulator of PKC and the calcium ionophore at the level of transcriptional activation in normal human T-cells are summarized in Fig. 1 to illustrate several consistent observations: (i) Highly purified normal human T-cells can be triggered to expressthe lymphokine genes,IL-2 and IFN--y, and the protooncogene, H-ras, with a synergistic combination of diCs and ionomycin, and independently of any additional antigenic or accessory cell signals (Fig. 1, lane 1 vs 4); (ii) diCS, at a concentration that fully activates the PKC (9) fails to induce gene expression in the absenceof an increase in intracellular free calcium concentration in T-cells (lane 3); and (iii) ionomycin, at a concentration that increases cytoplasmic free calcium concentration in normal hu-
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FIG. 1. Transcriptional activation by signaling T-cells with a synergistic combination of diCs and ionomycin. Highly purified T-cells were incubated alone, with ionomycin, with diCs, or with ionomycin and diC, in the presence and absence of cyclosporin A for 4 hr. RNA was isolated, and subjected to Northern blot analysis using several gene probes detecting activation genesof T lymphocytes. The blot was hybridized with cDNAs for (a) IL-Z (b) interferon-y, and (c) H-ras. Purified T-cells were treated as follows: lane 1, no treatment; lane 2, ionomycin; lane 3, diC8; lane 4, ionomycin and diCs ; and lanes 5-8, same experimental conditions as lanes l-4 + CsA ( 100 r&ml).
man T-cells from a resting level of - 100 nM to - 1000 nM (9), does not elicit maximal expression of lymphokine genesor the H-ras gene, and complementation of the calcium signal with the diCs signal is mandatory for gene expression in quiescent normal human T-cells as determined by Northern blot analysis (Fig. 1, lane 2 vs 4). In accordance with the transcription data shown in Fig. 1, neither diCs nor ionomycin induced marked T-cell proliferation, and combination of diCs and ionomycin was clearly synergistic in promoting new DNA synthesis in highly purified T-cells. In twelve experiments, [3H]thymidine incorporation (during 48-64 hr of culture) was 4388 -+ 1701 (mean + SEM, cpm/culture), 13,892 -t 3530,357s f 1338, and 71,120 + 12,527 when T-cells were incubated alone, ionomycin (1.0 P&Q alone, diC8 ( 10 pg/ml) alone, or with ionomycin (1 .O WV) and diCs (10 pg/ml), respectively. The proliferative response resulting from diCB and ionomycin was significant at P < 0.000 1 by analysis of variance.
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2
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FIG. 2. Analysis of low-level IL-2 transcription by polymerase chain reaction-amplified cDNA. RNA from T-cells stimulated with ionomycin and diCs was used to make cDNA with reverse transcriptase. The cDNA was amplified with Taq polymerase and Southern blotted for analysis with radiolabeled IL-2 cDNA. (Lane 1) Pooled cDNAs from T-cells treated for 1 hr or 4 hr with ionomycin; (lane 2) cells treated for 16 hr with ionomycin; (lane 3) pooled cDNAs from T-cells treated for 1 hr or 4 hr with diCs; (lane 4) cells treated for I6 hr with diCs; (lane 5) pooled cDNA from T-cells treated for 1 hr or 4 hr with ionomycin and diC,; (lane 6) cells treated for 16 hr with ionomycin and diCa. Polymerase chain reaction analysis of IL-2 cDNA for T-cells with no treatment for the I- plus 4-hr pool, and the 16-hr time point was negative for any IL-2 transcripts.
Analysis of Low-Level IL-2 Transcription by RT-PCR We used a powerful new methodology for mRNA analysis, sequential reverse transcription and polymerase chain reaction (RT-PCR), to supplement these initial Northern hybridization experiments. Moreover, this approach allowed us to establish differential gene induction capacity of diCs, ionomycin, or diCs and ionomycin in normal resting T-cells. As illustrated in Fig. 2, Southern hybridization with a 32P-IL2 cDNA probe of the RT-PCR product generated with IL-2 sequence-specific primer-pair and Taq polymerase revealed that: (i) signaling of T-cells with ionomycin alone results in only transient IL-2 gene expression despite continued presence of the calcium ionophore (Fig. 2, lane 1, T-cells incubated with ionomycin for 1 and 4 hr vs lane 2, T-cells incubated with ionomycin for 16 hr); (ii) diC8, alone, does not induce accumulation of IL-2 mRNA even when examined with the highly sensitive RT-PCR technique (lanes 3 and 4); and (iii) transmembrane signaling of T-cells with diCs and ionomycin clearly extends the duration of time during which IL-2 mRNA is transcribed and/or stabilized in normal human T-cells (Fig. 2, lanes 5 and 6). The PCR technique allowed us to analyze transcription data in experiments where low levels of IL-2 mRNA are synthesized in response to the ionomycin signal (compare Fig. 1, lane 2 with Fig. 2, lane 1). The RT-PCR technique shows that IL-2 mRNA exhibits an extended half-life and/or an increased transcription rate when it is synthesized in response to both signals rather than ionomycin alone (compare Fig. 2, lanes 1 and 2 vs lanes 5 and 6). This methodology allowed us to make this important obser-
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vation becauseit amplified low abundance IL-2 mRNA synthesis, not readily detectable by Northern blot analysis. Several explanations, not necessarily mutually exclusive, may explain the synergy between sn- 1,ZdiC* and ionomycin. First, ionomycin and diC8 might initiate independent cellular events that function as separatebut synergistic signals for gene transcription. Nishizuka (1) and others (13, 14) have shown that calcium ionophores induce phosphorylation of a 20-kDa protein in platelets, and TPA or synthetic diacylglycerols phosphorylate of a 40-kDa protein, and the physiological response of platelets is dependent upon signals generated with both the calcium ionophore and activators of PKC. Second, mobilization of calcium in T-cells with ionomycin might lead to the translocation of PKC from the cytosolic to membrane fraction, and increase the affinity of PKC for the diacylglycerols. Wolf et al. ( 16) have shown that the binding of purified PKC to the red cell membrane is dependent upon the concentration of calcium. May et al. ( 17) demonstrated that HL-60 human leukemic cells treated with the calcium ionophore A23 187 have increased affinity for phorbol esters.Third, in light of the recent demonstration of the presence of multiple and distinct forms of PKC ( 18) it is possible that differential requirements for calcium and diacylglycerols might exist among the family of PKC proteins. Indeed, the high degree of structural homology among calmodulin and a potential calcium binding domain in PKCa! is not conserved in PKCp or in PKC--r. Furthermore, synergism between diCs and ionomycin might be realized at the level of PKC activation itself as recently shown by Chatila et al. ( 15) in normal human T-cells stimulated with PMA and ionomycin. CsA Prevents IL-2 Gene Expression Elicited with diC8 and Ionomycin CsA’s effects on induced gene expression is dependent upon the nature of the activation signal and the responding cell type. It was of interest therefore to examine CsA’s effects on gene expression elicited with diCs , a physiologic regulator of PKC, and ionomycin. CsA suppressed transcription of IL-2, IFN-7 and H-r-as in T-cell cultures stimulated with ionomycin and diCs (Fig. 1, lanes 5-8). These results show that IL-2 transcription is inhibited by CsA and that CsA-induced suppression is not limited to lymphokine genes,but also affects G-protein (H-ras) transcription as well. CsA also decreased the expression of pyruvate kinase mRNA expression in ionomycin/diC*-stimulated T-cells, as well as abrogated T-cell proliferation (data not shown). Addition of cycloheximide to cultures also containing CsA did not facilitate amplified steady-state levels of IL-2 mRNA. Although some T-cell activation genes are destabilized by a cyclohexamide-sensitive mechanism, RNA destabilization is not the primary mechanism explaining CsA’s ability to decrease cytoplasmic levels of certain T-cell activation transcripts. Analysis of RT-PCR products generated with IL-2 or fl actin (control for constitutive mRNA) sequence-specific oligomers was performed. Transmembrane signaling of normal human T-cells with diCg and ionomycin stimulated IL-2 gene activation in normal human T-cells (Fig. 3, lane 2 vs 4); CsA, however, prevented diCs and ionomycin-dependent IL-2 gene expression (Fig. 3, lane 4 or 6). Constitutive /3actin gene expression was not affected by T-cell activation, and/or by CsA (Fig. 3, lanes 1, 3, and 5). The DNA fragments shown in Fig. 3 are consistent with expected sizes based on the oligomer selection for the IL-2 and actin genes.
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s123456S
492-369-246--
Actin
123-o
FIG. 3. Determination of GA’s effects with the RT-PCR. Total cellular RNA was isolated, reverse transcribed with oligo (dT) primers and M-MLV reverse transcriptase. The first-strand cDNA was amplified with either IL-2 sequence-specific or fl actin sequence-specific primer pair and Taq polymerase. The amplified PCR products were fractionated by agarosegel electrophoresis, stained with ethidium bromide, and validated by the predicted size of IL-2 cDNA fragments (120 base pairs) or @-actincDNA fragments (349 base pairs). Lanes 1 and 2: T-cells incubated alone for 16 hr, lanes 3 and 4: T-cells + diC, (10.0 ag/ ml) + ionomycin ( I .OPM), and lanes 5 and 6: T + CsA (100 &ml) + diCs and ionomycin. Cycloheximide (20.0 pg/ml) was added 4 hr after initiation of T-cell cultures. (S) lanes on the left and right indicate marker lanes.
CsA Prevents Gene Expression by a Direct Eflect on Normal Human T-Cells CsA’s multiple effectson accessorycells has lead to the argument that CsA prevents T-cell activation via its effects on accessory cells ( 19-22). However, our demonstration that CsA prevents IL-2 gene expression elicited with diCB and ionomycin indicates that CsA directly inhibits IL-2 gene expression in normal human T-cells, independent of its effects on accessorycells. In order to confirm and extend this observation, we determined CsA’s effects on accessory cell-independent gene expression induced by crosslinking the CD3 and CD2 antigens on the T-cell surface. The proliferative response is dependent upon signaling T-cells with both crosslinked anti-CD3 and anti-CD2 mAbs, and is independent of accessory cells since crosslinked antiCD2 functions essentially as an accessorycell substitute. Northern blot analyses demonstrating CsA’s inhibitory effect on accessory cellindependent gene induction in normal human T-cells are summarized in Fig. 4. It is
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IJ.-2
FIG. 4. Confirmation of CsA’s direct inhibitory effect on normal human T-cells: T-cells were incubated alone (lane 1); with crosslinked anti-CD2 (OKTI I, 0.5 &ml, lane 2); crosslinked anti-CD3 (OKT3, 0.5 Ng/ml, lane 3); crosslinked anti-CD2 and anti-CD3 (lane 4); or crosslinked anti-CD2 and anti-CD3 plus CsA (100 rig/ml) for 6 hr, (lane 5). RNA was then isolated and analyzed for (a) IL-2 and (b) IFN--, mRNA by Northern hybridization.
evident that crosslinking of CD2 and CD3 proteins on the T-cell surface with antiCD3 and anti-CD2 mAbs results in activation of the IL-2 gene in highly purified normal human quiescent T-cells (Figure 4a, lane 4). CsA prevents anti-CD2 and antiCD3 mAb signal-dependent IL-2 mRNA accumulation (Fig. 4a, lane 5). This mode of T-cell activation also promoted IFN--r transcription (Fig. 4b, lane 4), while CsA prevented IFN-7 mRNA accumulation induced with the synergistic combination of mAbs directed at the CD2 and CD3 antigens (Fig. 4b, lane 5). Crosslinking the CD2 antigen alone or CD3 antigen alone is insufficient for promoting IL-2 or IFN--/ transcription (Figs. 4a and 4b, lanes 2 and 3). Investigation of DNA synthesis in T-cells demonstrated the requirement of crosslinking CD2 and CD3 antigen on the T-cell surface. In nine experiments, [3H]thymidine incorporation was 1298 + 445 (mean + SEM, cpm/culture), 399 1 f 1324, 2201 & 1151, or 72291 + 9380 cpm/culture when T-cells were incubated alone, crosslinked anti-CD3 mAbs, crosslinked antiCD2 mAbs, or crosslinked anti-CD3 + anti-CD2 mAbs, respectively. The prolifera-
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tive response occurring as a consequence of signaling T-cells via the CD3 and CD2 antigen was significant at P < 0.000 1 by analysis of variance. Our gene transcription data supports the work of others showing the presence of accessorycell signals is obligatory for ionomycin-induced proliferation of purified Tcells (9, 15). We observed very low levels of IL-2 transcription by Northern blot analysis in T-cells cultured with ionomycin alone; transcription was greatly enhanced with addition of a PKC activator, diC8. Furthermore, our results show a superinduction of IL-2 and IFN-7 transcription when PMA was present in the culture with ionomycin (data not shown), a level much greater than T-cell cultures containing ionomycin and diC8. Our gene transcription results are consistent with prior reports concerning the synergy between ionomycin and diC8 (23), but differ from others (24). The controversy over whether ionomycin and DAG analogs synergize to promote Tcell activation could be explained by differences in cell culture conditions or the manner of preparation of the cells and reagents. Each reagent, added separately, was clearly ineffective in promoting transcription, but together caused a large increase in both cell proliferation and gene transcription. Our inclusion of the anti-CD2/antiCD3 model of T-cell activation also addressesthis issue. Again, each stimulus alone was completely ineffective in promoting T-cell proliferation (25) and gene transcription, but together caused transcription and proliferation. These T-cell activation paradigms act through different cell surface pathways in an accessory cell-independent fashion, but both require two signals, one of which activates PKC. Furthermore, we find significant levels of IL-2 receptor expression (Tat) in both activation models. The kinetics of synthesis of the Tat mRNA is clearly very different, and more elusive, than when phorbol esters are employed (manuscript in preparation). The inhibitory effects of CsA on proliferation (26) and gene transcription in these models is consistent with the performance of CsA as a reagent which blocks IL-2 transcription and cell proliferation. In summary, results from the current investigation demonstrate for the first time that a physiologic regulator of PKC, diC8 or crosslinking of the CD2 and CD3 antigens on the T-cell surface results in the transcription of growth-promoting genes in normal human resting T-cells. Using the powerful RT-PCR technique we were able to assign specific roles for ionomycin, diCs , or the combination in activation of specific T-cell activation genes. Our studies also reveal that CsA, a clinically useful immunosuppressant, prevents gene expression by a direct effect on normal human quiescent T-cells, independent of its effects on accessory cells. These observations are especially significant in light of the experimental conditions used in this investigation-utilization of normal rather than human T-cell lines, and T-cell signaling with physiologic rather than nonphysiologic regulators of PKC, as well as use of an antigen-dependent T-cell activation pathway (CD2-CD3 crosslinking) rather than with plant lectins. REFERENCES 1. 2. 3. 4. 5. 6.
Nishizuka, Y., Nature (London) 308,693, 1984. Kaibuchi, K., Takai, Y., and Nishizuka, Y., J. Biol. Chem. 260, 1366, 1985. Delia, D., &eaves, M., Villa, S., and DeBraud, F., Eur. J. Immunok 14,720, 1984. Truneh, A., Albert, F., Golstein, P., and Schmitt-Verhulst, A. M., Nature (London) 313,3 18, 1985. Truneh, A., Albert, F., Golstein, P., and Schmitt-Verhulst, A. M., J. Immunol. 135,2262, 1985. Yamamoto, S., Gotoh, S., Aizu, E., and Kato, R., J. Biol. Chem. 260, 14,230, 1985.
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7. Kaibuchi, K., Sano, K., Hoshijima, M., Takai, Y., and Nishizuka, Y., Cell Calcium. 3,323, 1982. 8. May, W. S., Jr., Lapetina, E. G., and Cuatrecasas, P., Proc. Natl. Acad. Sci. USA 83, 1281, 1986. 9. Subramaniam, P., Sehajpal, P., Murthi, V. K., Stenzel, K. H., and Suthanthiran, M., Cell. Immunol. 116,439, 1988. IO. Chirgwin, J., Pryzbyla, A., MacDonald, R., and Rutter, W., Biochemistry. l&5294, 1978. 11. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H., Biochemistry. 16,4743, 1977. 12. Feinberg, A. P.. and Vogelstein, B., Analytical Biochem. 132,6, 1983. 13. Lapentina, E. G., Reep, B., Ganong, B. R., and Bell, R. M., J. Biol. Chem. 260, 1358, 1985. 14. Davies, R. J., Ganong, B. R., Bell, R. M., and Czech, M. P., J. Biol. Chem. 260,53 15, 1985. 15. Chatila, T., Silverman, L., Miller, R., and Geha, R., J. Immunol. 143, 1283, 1989. 16. Wolf, M., Levine, H., III, May, W. S., Cuatrecasas, P., and Sahyoun, N., Nature (London) 317, 546, 1985. 17. May, W. S., Jr., Sahyoun, N., Wolf, M., and Cuatrecasas, P., Nature (London) 317,549, 1985. 18. Coussens, L., Parker, P. J., Rhee, L., Yang-Feng, T. L., Chen, E., Waterfield, M. D., Francke, V., and Ullvain, A., Science 233,859, 1986. 19. Palacios, R., J. Immunol. 128,337, 1982. 20. Hess, A. D., and Colomboni, P. M., Transplant. Proc. 18,219, 1986. 21. Uyemura, K., Dixon, J. F. P., and Parker, J. W., Transplant. Proc. l&2376, 1983. 22. Manta, F., Kunkl, A., and Celada, F., Transplantation. 39,644, 1985. 23. Manger, B., Weiss, A., Imboden, J., Laing, T., and Stobo, J. D., J. Immunol. 139,2755, 1987. 24. Berry, N., Ase, K., Kikkawa, U., Kishimoto, A., and Nishizuka, Y., J. Immunol. 143, 1407, 1989. 25. Suthanthiran, M., Cell. Immunol. 112, 112, 1988. 26. Sehajpal, P., Subramaniam, A., Murthi, V. K., Sharma, B. K., and Suthanthiran, M., Cell. Immunol. 120, 195, 1989.
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Physiologic Signaling in Normal Human T-cells: mRNA Phenotyping by Northern Blot Analysis and Reverse TranscriptionPolymerase Chain Reaction’ KENNETHJ. WIEDER,*GERDWALZ,*BERND ZANKER,* PRABODHSEHAJPAL, VIJAY K. SHARMA,EDWARDSKOLNIK,TERRY B. STROM,* AND MANIKKAM~UTHANTHIRAN *Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215 and The Rogosin Institute, Department ofMedicine and Biochemistry, Cornell University Medical College, New York, New York 10021 Received November IO. 1989; accepted January 16, 1990
Synergy between ionomycin and sn- l,2-dioctanoylglycerol (diCs) was shown at the level of lymphokine gene transcription. Transcriptional activation of interleukin-2 (IL-2), interferon-r (EN-r), and the protooncogene H-ras was accomplished by signaling highly purified normal human resting T-lymphocytes (T-cells) with diCs, a physiologic regulator of protein kinase C, and the calcium ionophore, ionomycin. Northern blot analysis of mRNA for early T-cell activation genesdemonstrated the synergism between diCs and ionomycin at the gene induction level. To amplify very low levels of IL-2 mRNA, sequential reverse transcription and polymerase chain reaction (RT-PCR) of T cell mRNA were used to demonstrate the capacity ofthe calcium signal (ionomycin) to promote low-level IL-2 transcription in normal human T-cells without additional signals. Cyclosporine (CsA) prevented diCs and ionomycin-induced expression of IL2, IFN-7, and H-ras genes. The completeness of its inhibitory effect was evident by the absence of IL-2 transcripts in CsA-treated cultures screened by the RT-PCR technique. CsA also prevented IL-2 and IF%-, gene expression in accessory cell-depleted T-cells stimulated by crosslinking the CD2 and CD3 antigens on the cell surface. Our observations demonstrate that a physiologic regulator of PKC, diCs, and cell surface crosslinking of the CD2 and CD3 antigen, promote gene expression in normal human quiescent T-cells independently of accessorycells, and that CsA prevents gene expression via a direct effect on T-cells. o 1990 Academic PXSS, IX.
INTRODUCTION An increase in the concentration of intracellular free calcium and the activation of protein kinase C (PKC) are thought to function as synergistic intracellular signals for the induced expression of growth-promoting genesin T-cells (1,2). However, much of the data that support this unifying theme for transcriptional activation have come largely from studies where preactivated T-cells (e.g., T-cell clones, T-leukemic lines etc.) rather than normal T-cells and nonphysiologic rather than physiologic activators of PKC have been utilized (3-5). Because the signaling requirements for normal, ’ This work was supported by Grants ROl-CA43977 and ROl-DK32929 (K.J.W., G.W., B.Z., and T.B.S.) from the National Institute of Health. 41 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
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primary T-cell activation may differ from leukemic or preactivated T-cells (6), including the documented dissimilarities between the nonphysiologic regulators of PKC such as phorbol estersand the physiologic diacylglycerols ( 1,7,8), additional studies were undertaken. In this report we sought to explore basic mechanisms concerning T-cell activation using the physiological activators of T-cells, ionomycin and diCs, and CD2/CD3 crosslinking, at the level of lymphokine and protooncogene transcription. Stimulation of highly purified T-cells with ionomycin, an accessorycell-dependent event, is augmented by physiologic activators of PKC (9). Since ionomycin and PKC activators are both required to stimulate T-cell activation, we wondered whether ionomycin activates one set of T-cell activation genesand another complementary set is activated by PKC activators or whether both signals are required to activate these genes. Similar information was sought in a system that crosslinked CD2-CD3 proteins with monoclonal antibodies (mAbs). Gene transcription data gathered by Northern blot analysis was supplemented by use of the polymerasechain reaction which allowed us to detect low abundance IL-2 transcription which was not obvious by Northern blot analysis. Here we report that synthetic and membrane permeant sn- 1,2-dioctanoylglycerol (diC*) transcriptionally activates interleukin-2 (IL-2) interferon-y (IFN-7) and H-ras genesin T-cells in the presence of ionomycin, independent of additional antigenic or accessorycell signals. Our results also show that cyclosporine (CsA), a clinically useful drug, prevents gene expression induced with the synergistic combination of diCs and ionomycin by a direct effect on T-cells. This new observation is extended by our additional demonstration that CsA prevents gene expression elicited by signaling of T-cells via the CD2 and CD3 antigens-an antigen-dependent T-cell activation pathway where the calcium ions and PKC activation contribute to an intracytoplasmic signal transduction pathway. MATERIALS AND METHODS T-Cell Isolation Human peripheral blood mononuclear cells (PBMCs) were isolated by FicollPaque density gradient centrifugation of venous blood obtained from healthy adult volunteers. T-cells were isolated from the PBMCs with a slight modification of a sequential four-step procedure which includes: (i) depletion of adherent cells by Sephadex G- 10 gel filtration, (ii) depletion of residual monocytes by treatment with 5 mM leucine methyl ester treatment, (iii) depletion of HLA-DR bearing cells and natural killer cells by treatment with anti-HLA-DR mAbs directed at the monomorphic component of HLA-DR antigen, 28-34 kDa), anti-NKH-1 A mAb (defining a 200-kDa molecule expressed on all NK cells but not abundantly expressedby other lymphoid or myeloid cells), and prescreened rabbit complement, and (iv) utilization of the SRBC-rosetting technique as the final preparative step for the isolation of T-cells. Fluorescence-activated cell sorter phenotypic analyses of a representative T-cell preparation: 99% CD2+, 98% CD3+, 0.36% NKH-I+, 0.34% HLA-DR+, 0.07% Leu-M3 (CD14+), 0.05% Leu-12 (CD19+), and 0.56% IL-2R (CD25+) cells. Activation with diC, and Ionomycin T-cells were ( lo6 cells/ml) suspended in RPM1 1640 medium supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin, 25 mM Hepes buffer, and 5% heat-
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inactivated fetal bovine serum (complete medium), and were incubated with predetermined optimum concentrations of diCs (10.0 pg/ml) and ionomycin (1 .O mM). The proliferative response is dependent upon signaling T-cells with both ionomycin and DAG, and is independent of additional antigenic or accessorysignals (9). Ionomycin (Streptomyces conglobatus [ATCC 3 1005]) was purchased from Calbiochem Biochemicals (San Diego, CA). DiCs was purchased from Sigma Chemical Co. (St. Louis, MO). Ionomycin and diC8 were initially dissolved in ethyl alcohol, and phosphate-buffered saline (PBS, Sigma) was then used to dilute the stock solution to the appropriate concentrations of each agent. T-cell Activation with Crosslinked Anti-CD2 and Anti-CD3 mAbs T-cells ( 1O6cells/ml) suspended in complete medium were incubated with 0.5 pug/ ml of anti-CD3 mAb antibodies (OKT3, Ortho Diagnostics Systems, Inc., Raritan, NJ), 0.5 lug/ml of anti-CD2 mAb (OKTll, Ortho Diagnostics) and 5.0 pg/ml of affinity-purified goat antibodies specific for mouse IgG (Southern Biotechnology Associates, Birmingham, AL). Polymerase Chain Reaction (PCR) PCR was employed to detect low abundance mRNA encoding IL-2 or actin in stimulated T cells. A 32-mer (“antisense”) oligonucleotide (5’-GGCAGAAGCTTGGCCTGATATGTTTTAAGTGGG-3’), 22 of which are complementary to the 3’ noncoding region of the IL-2 cDNA, and a second 26-mer (“sense”) oligonucleotide (5’-CAGTGTCTAGAAGAAGAACTCAAACC-3’) homologous to the IL-2 coding region at the XbaI restriction site were synthesized on an Applied Biosystems 38 1A DNA synthesizer. Actin oligomers were also synthesized which would amplify a 349basepair fragment of the actin transcript as described below. First-strand cDNA copies of the IL-2 and actin mRNAs were synthesized in a I O-p1reaction volume containing 10-pg of total RNA, 0.5 PM antisense oligonucleotide, and 200 units of M-MLV reverse transcriptase (Bethesda Research Laboratories, Bethesda, MD). The entire cDNA synthesis reaction volume was then combined in a 50-~1final reaction volume for PCR amplification containing 0.25 PMeach oligonucleotide primer, and I .5 units of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) under conditions suggested by the manufacturer. Forty cycles of PCR amplification were performed using a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). Each cycle consisted of 45 set for denaturation at 94°C 30 set for annealing at 55°C and 30 set for enzymatic primer extension at 72°C. Specificity of the amplified IL-2 and actin cDNAs fragments were determined by Southern blot analysis utilizing the IL-2 full-length cDNA, or identification of the appropriately sized ethidium bromide stained DNA fragment. In some experiments, first-strand cDNA copies were synthesized using oligo (dT) primers and M-MLV reverse transcriptase, and were then amplified with a sequencespecific primer pair using Taq polymerase. The PCR-generated products were then analyzed by agarosegel electrophoresis for the predicted size of cDNA fragment. RNA Isolation and Northern Blot Analysis Total RNA from T-cell preparations was isolated by the guanidinium isothiocyanate/cesium chloride method (10). Twenty micrograms of total RNA was run on a
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2.2 M formaldehyde/l% agarose gel (11). The gel was blotted directly onto Amersham’s Hybond nylon in 20X SSC(3.0 M NaC1/0.3 M Na citrate, pH 7). RNA transferred onto the filter was covalently immobilized to the support by exposure to 302 nm UV light for 4 min. The filter was prehybridized for 5 hr, then hybridized at 42°C to cDNA probes in a solution containing 50 mM sodium phosphate, pH 6.5, 50% formamide, 0.1% SDS, 8% dextran sulfate, 100 pg/ml denatured salmon sperm DNA, 3X Denhardt’s solution, 5X SSC. Following hybridization for 24 hr filters were washed twice in 0.2X SSC/O.1%SDS at room temperature for 10 min each, and twice at 55°C using the same solution for 20 min each. Filters are placed against Agfa RPI film for l-3 days. DNA Probes and Labeling IL-2 cDNA was provided by N. Farber (Biogen, Inc., Cambridge, MA) H-ras cDNA was obtained from ATCC, IL-2 receptor (Tat) cDNA was provided by Dr. Warren Leonard (National Institute of Health, Bethesda, MD), and interferon-y cDNA was provided by Dr. Steven Clark (Genetics Institute, Cambridge, MA). Complementary DNA was isolated from their plasmid vectors by restriction enzyme digestion and electroelution from agarosegel slices. The cDNAs were labeled with [32P]dCTP using the random priming procedure (12). Labeled cDNAs were added to hybridization solution at a concentration of 1 X lo6 cpm/ml of solution. RESULTS AND DISCUSSION Synergistic Transmembrane Signaling of T-Cells with die, and Ionomycin Diacylglycerols possessing eight carbon atom acyl chains are potent stimulators of PKC with the 3 hydroxyl group being essential for the successful interaction of diacylglycerol with PKC ( 13, 14). We therefore utilized diCs, a physiologic analogue of DAG, to activate PKC in highly purified normal human T-cells. Signaling of Tcells with diCs was accomplished in the presence of ionomycin, a highly specific calcium ionophore, because of the demonstrated synergy between the calcium signal and PKC activation for the elicitation of T-cell proliferation (9). The basis for this synergy has recently been explored in a thorough study of the signal transducing mechanisms that are activated by ionomycin or PMA, or both, in highly purified Tcells ( 15). Nonetheless this system has not been thoroughly explored at the transcriptional level. Moreover, we have utilized diCs rather than PMA because it is a physiological analog of diacylglycerol which also can activate PKC, without additional effectsexerted by phorbol esters(6, 14). Northern blot analysesdemonstrating the synergism between the physiological regulator of PKC and the calcium ionophore at the level of transcriptional activation in normal human T-cells are summarized in Fig. 1 to illustrate several consistent observations: (i) Highly purified normal human T-cells can be triggered to expressthe lymphokine genes,IL-2 and IFN--y, and the protooncogene, H-ras, with a synergistic combination of diCs and ionomycin, and independently of any additional antigenic or accessory cell signals (Fig. 1, lane 1 vs 4); (ii) diCS, at a concentration that fully activates the PKC (9) fails to induce gene expression in the absenceof an increase in intracellular free calcium concentration in T-cells (lane 3); and (iii) ionomycin, at a concentration that increases cytoplasmic free calcium concentration in normal hu-
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FIG. 1. Transcriptional activation by signaling T-cells with a synergistic combination of diCs and ionomycin. Highly purified T-cells were incubated alone, with ionomycin, with diCs, or with ionomycin and diC, in the presence and absence of cyclosporin A for 4 hr. RNA was isolated, and subjected to Northern blot analysis using several gene probes detecting activation genesof T lymphocytes. The blot was hybridized with cDNAs for (a) IL-Z (b) interferon-y, and (c) H-ras. Purified T-cells were treated as follows: lane 1, no treatment; lane 2, ionomycin; lane 3, diC8; lane 4, ionomycin and diCs ; and lanes 5-8, same experimental conditions as lanes l-4 + CsA ( 100 r&ml).
man T-cells from a resting level of - 100 nM to - 1000 nM (9), does not elicit maximal expression of lymphokine genesor the H-ras gene, and complementation of the calcium signal with the diCs signal is mandatory for gene expression in quiescent normal human T-cells as determined by Northern blot analysis (Fig. 1, lane 2 vs 4). In accordance with the transcription data shown in Fig. 1, neither diCs nor ionomycin induced marked T-cell proliferation, and combination of diCs and ionomycin was clearly synergistic in promoting new DNA synthesis in highly purified T-cells. In twelve experiments, [3H]thymidine incorporation (during 48-64 hr of culture) was 4388 -+ 1701 (mean + SEM, cpm/culture), 13,892 -t 3530,357s f 1338, and 71,120 + 12,527 when T-cells were incubated alone, ionomycin (1.0 P&Q alone, diC8 ( 10 pg/ml) alone, or with ionomycin (1 .O WV) and diCs (10 pg/ml), respectively. The proliferative response resulting from diCB and ionomycin was significant at P < 0.000 1 by analysis of variance.
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FIG. 2. Analysis of low-level IL-2 transcription by polymerase chain reaction-amplified cDNA. RNA from T-cells stimulated with ionomycin and diCs was used to make cDNA with reverse transcriptase. The cDNA was amplified with Taq polymerase and Southern blotted for analysis with radiolabeled IL-2 cDNA. (Lane 1) Pooled cDNAs from T-cells treated for 1 hr or 4 hr with ionomycin; (lane 2) cells treated for 16 hr with ionomycin; (lane 3) pooled cDNAs from T-cells treated for 1 hr or 4 hr with diCs; (lane 4) cells treated for I6 hr with diCs; (lane 5) pooled cDNA from T-cells treated for 1 hr or 4 hr with ionomycin and diC,; (lane 6) cells treated for 16 hr with ionomycin and diCa. Polymerase chain reaction analysis of IL-2 cDNA for T-cells with no treatment for the I- plus 4-hr pool, and the 16-hr time point was negative for any IL-2 transcripts.
Analysis of Low-Level IL-2 Transcription by RT-PCR We used a powerful new methodology for mRNA analysis, sequential reverse transcription and polymerase chain reaction (RT-PCR), to supplement these initial Northern hybridization experiments. Moreover, this approach allowed us to establish differential gene induction capacity of diCs, ionomycin, or diCs and ionomycin in normal resting T-cells. As illustrated in Fig. 2, Southern hybridization with a 32P-IL2 cDNA probe of the RT-PCR product generated with IL-2 sequence-specific primer-pair and Taq polymerase revealed that: (i) signaling of T-cells with ionomycin alone results in only transient IL-2 gene expression despite continued presence of the calcium ionophore (Fig. 2, lane 1, T-cells incubated with ionomycin for 1 and 4 hr vs lane 2, T-cells incubated with ionomycin for 16 hr); (ii) diC8, alone, does not induce accumulation of IL-2 mRNA even when examined with the highly sensitive RT-PCR technique (lanes 3 and 4); and (iii) transmembrane signaling of T-cells with diCs and ionomycin clearly extends the duration of time during which IL-2 mRNA is transcribed and/or stabilized in normal human T-cells (Fig. 2, lanes 5 and 6). The PCR technique allowed us to analyze transcription data in experiments where low levels of IL-2 mRNA are synthesized in response to the ionomycin signal (compare Fig. 1, lane 2 with Fig. 2, lane 1). The RT-PCR technique shows that IL-2 mRNA exhibits an extended half-life and/or an increased transcription rate when it is synthesized in response to both signals rather than ionomycin alone (compare Fig. 2, lanes 1 and 2 vs lanes 5 and 6). This methodology allowed us to make this important obser-
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vation becauseit amplified low abundance IL-2 mRNA synthesis, not readily detectable by Northern blot analysis. Several explanations, not necessarily mutually exclusive, may explain the synergy between sn- 1,ZdiC* and ionomycin. First, ionomycin and diC8 might initiate independent cellular events that function as separatebut synergistic signals for gene transcription. Nishizuka (1) and others (13, 14) have shown that calcium ionophores induce phosphorylation of a 20-kDa protein in platelets, and TPA or synthetic diacylglycerols phosphorylate of a 40-kDa protein, and the physiological response of platelets is dependent upon signals generated with both the calcium ionophore and activators of PKC. Second, mobilization of calcium in T-cells with ionomycin might lead to the translocation of PKC from the cytosolic to membrane fraction, and increase the affinity of PKC for the diacylglycerols. Wolf et al. ( 16) have shown that the binding of purified PKC to the red cell membrane is dependent upon the concentration of calcium. May et al. ( 17) demonstrated that HL-60 human leukemic cells treated with the calcium ionophore A23 187 have increased affinity for phorbol esters.Third, in light of the recent demonstration of the presence of multiple and distinct forms of PKC ( 18) it is possible that differential requirements for calcium and diacylglycerols might exist among the family of PKC proteins. Indeed, the high degree of structural homology among calmodulin and a potential calcium binding domain in PKCa! is not conserved in PKCp or in PKC--r. Furthermore, synergism between diCs and ionomycin might be realized at the level of PKC activation itself as recently shown by Chatila et al. ( 15) in normal human T-cells stimulated with PMA and ionomycin. CsA Prevents IL-2 Gene Expression Elicited with diC8 and Ionomycin CsA’s effects on induced gene expression is dependent upon the nature of the activation signal and the responding cell type. It was of interest therefore to examine CsA’s effects on gene expression elicited with diCs , a physiologic regulator of PKC, and ionomycin. CsA suppressed transcription of IL-2, IFN-7 and H-r-as in T-cell cultures stimulated with ionomycin and diCs (Fig. 1, lanes 5-8). These results show that IL-2 transcription is inhibited by CsA and that CsA-induced suppression is not limited to lymphokine genes,but also affects G-protein (H-ras) transcription as well. CsA also decreased the expression of pyruvate kinase mRNA expression in ionomycin/diC*-stimulated T-cells, as well as abrogated T-cell proliferation (data not shown). Addition of cycloheximide to cultures also containing CsA did not facilitate amplified steady-state levels of IL-2 mRNA. Although some T-cell activation genes are destabilized by a cyclohexamide-sensitive mechanism, RNA destabilization is not the primary mechanism explaining CsA’s ability to decrease cytoplasmic levels of certain T-cell activation transcripts. Analysis of RT-PCR products generated with IL-2 or fl actin (control for constitutive mRNA) sequence-specific oligomers was performed. Transmembrane signaling of normal human T-cells with diCg and ionomycin stimulated IL-2 gene activation in normal human T-cells (Fig. 3, lane 2 vs 4); CsA, however, prevented diCs and ionomycin-dependent IL-2 gene expression (Fig. 3, lane 4 or 6). Constitutive /3actin gene expression was not affected by T-cell activation, and/or by CsA (Fig. 3, lanes 1, 3, and 5). The DNA fragments shown in Fig. 3 are consistent with expected sizes based on the oligomer selection for the IL-2 and actin genes.
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s123456S
492-369-246--
Actin
123-o
FIG. 3. Determination of GA’s effects with the RT-PCR. Total cellular RNA was isolated, reverse transcribed with oligo (dT) primers and M-MLV reverse transcriptase. The first-strand cDNA was amplified with either IL-2 sequence-specific or fl actin sequence-specific primer pair and Taq polymerase. The amplified PCR products were fractionated by agarosegel electrophoresis, stained with ethidium bromide, and validated by the predicted size of IL-2 cDNA fragments (120 base pairs) or @-actincDNA fragments (349 base pairs). Lanes 1 and 2: T-cells incubated alone for 16 hr, lanes 3 and 4: T-cells + diC, (10.0 ag/ ml) + ionomycin ( I .OPM), and lanes 5 and 6: T + CsA (100 &ml) + diCs and ionomycin. Cycloheximide (20.0 pg/ml) was added 4 hr after initiation of T-cell cultures. (S) lanes on the left and right indicate marker lanes.
CsA Prevents Gene Expression by a Direct Eflect on Normal Human T-Cells CsA’s multiple effectson accessorycells has lead to the argument that CsA prevents T-cell activation via its effects on accessory cells ( 19-22). However, our demonstration that CsA prevents IL-2 gene expression elicited with diCB and ionomycin indicates that CsA directly inhibits IL-2 gene expression in normal human T-cells, independent of its effects on accessorycells. In order to confirm and extend this observation, we determined CsA’s effects on accessory cell-independent gene expression induced by crosslinking the CD3 and CD2 antigens on the T-cell surface. The proliferative response is dependent upon signaling T-cells with both crosslinked anti-CD3 and anti-CD2 mAbs, and is independent of accessory cells since crosslinked antiCD2 functions essentially as an accessorycell substitute. Northern blot analyses demonstrating CsA’s inhibitory effect on accessory cellindependent gene induction in normal human T-cells are summarized in Fig. 4. It is
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IJ.-2
FIG. 4. Confirmation of CsA’s direct inhibitory effect on normal human T-cells: T-cells were incubated alone (lane 1); with crosslinked anti-CD2 (OKTI I, 0.5 &ml, lane 2); crosslinked anti-CD3 (OKT3, 0.5 Ng/ml, lane 3); crosslinked anti-CD2 and anti-CD3 (lane 4); or crosslinked anti-CD2 and anti-CD3 plus CsA (100 rig/ml) for 6 hr, (lane 5). RNA was then isolated and analyzed for (a) IL-2 and (b) IFN--, mRNA by Northern hybridization.
evident that crosslinking of CD2 and CD3 proteins on the T-cell surface with antiCD3 and anti-CD2 mAbs results in activation of the IL-2 gene in highly purified normal human quiescent T-cells (Figure 4a, lane 4). CsA prevents anti-CD2 and antiCD3 mAb signal-dependent IL-2 mRNA accumulation (Fig. 4a, lane 5). This mode of T-cell activation also promoted IFN--r transcription (Fig. 4b, lane 4), while CsA prevented IFN-7 mRNA accumulation induced with the synergistic combination of mAbs directed at the CD2 and CD3 antigens (Fig. 4b, lane 5). Crosslinking the CD2 antigen alone or CD3 antigen alone is insufficient for promoting IL-2 or IFN--/ transcription (Figs. 4a and 4b, lanes 2 and 3). Investigation of DNA synthesis in T-cells demonstrated the requirement of crosslinking CD2 and CD3 antigen on the T-cell surface. In nine experiments, [3H]thymidine incorporation was 1298 + 445 (mean + SEM, cpm/culture), 399 1 f 1324, 2201 & 1151, or 72291 + 9380 cpm/culture when T-cells were incubated alone, crosslinked anti-CD3 mAbs, crosslinked antiCD2 mAbs, or crosslinked anti-CD3 + anti-CD2 mAbs, respectively. The prolifera-
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tive response occurring as a consequence of signaling T-cells via the CD3 and CD2 antigen was significant at P < 0.000 1 by analysis of variance. Our gene transcription data supports the work of others showing the presence of accessorycell signals is obligatory for ionomycin-induced proliferation of purified Tcells (9, 15). We observed very low levels of IL-2 transcription by Northern blot analysis in T-cells cultured with ionomycin alone; transcription was greatly enhanced with addition of a PKC activator, diC8. Furthermore, our results show a superinduction of IL-2 and IFN-7 transcription when PMA was present in the culture with ionomycin (data not shown), a level much greater than T-cell cultures containing ionomycin and diC8. Our gene transcription results are consistent with prior reports concerning the synergy between ionomycin and diC8 (23), but differ from others (24). The controversy over whether ionomycin and DAG analogs synergize to promote Tcell activation could be explained by differences in cell culture conditions or the manner of preparation of the cells and reagents. Each reagent, added separately, was clearly ineffective in promoting transcription, but together caused a large increase in both cell proliferation and gene transcription. Our inclusion of the anti-CD2/antiCD3 model of T-cell activation also addressesthis issue. Again, each stimulus alone was completely ineffective in promoting T-cell proliferation (25) and gene transcription, but together caused transcription and proliferation. These T-cell activation paradigms act through different cell surface pathways in an accessory cell-independent fashion, but both require two signals, one of which activates PKC. Furthermore, we find significant levels of IL-2 receptor expression (Tat) in both activation models. The kinetics of synthesis of the Tat mRNA is clearly very different, and more elusive, than when phorbol esters are employed (manuscript in preparation). The inhibitory effects of CsA on proliferation (26) and gene transcription in these models is consistent with the performance of CsA as a reagent which blocks IL-2 transcription and cell proliferation. In summary, results from the current investigation demonstrate for the first time that a physiologic regulator of PKC, diC8 or crosslinking of the CD2 and CD3 antigens on the T-cell surface results in the transcription of growth-promoting genes in normal human resting T-cells. Using the powerful RT-PCR technique we were able to assign specific roles for ionomycin, diCs , or the combination in activation of specific T-cell activation genes. Our studies also reveal that CsA, a clinically useful immunosuppressant, prevents gene expression by a direct effect on normal human quiescent T-cells, independent of its effects on accessory cells. These observations are especially significant in light of the experimental conditions used in this investigation-utilization of normal rather than human T-cell lines, and T-cell signaling with physiologic rather than nonphysiologic regulators of PKC, as well as use of an antigen-dependent T-cell activation pathway (CD2-CD3 crosslinking) rather than with plant lectins. REFERENCES 1. 2. 3. 4. 5. 6.
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