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House set to hold override vote on Trump's first veto
by: James Selby
Posted: Mar 26, 2019 / 11:22 AM CDT / Updated: Mar 26, 2019 / 11:22 AM CDT
(3/26/19) The House will vote Tuesday whether to override the President's first veto in a largely symbolic effort that reflects the new era of divided government under President Donald Trump.
It's a steep hill to climb for opponents of Trump's national emergency as the House would need two-thirds of its members to back the veto override.
Both chambers of Congress passed a resolution to overturn Trump's national emergency declaration to fund more border wall, which would use billions from the Treasury and Defense departments after Congress rejected giving Trump the full amount of border wall money he requested for the year.
The decision sparked criticism from Republicans and Democrats alike, many of whom argued the national emergency action would violate the Constitution.
The House easily passed the resolution of disapproval in February. A few weeks later, 12 Senate Republicans joined with Senate Democrats to pass it, marking a sharp rebuke from members of the President's own party on a key issue to his base.
The President vetoed the bill one day after it cleared the Senate.
"Congress has the freedom to pass this resolution and I have the duty to veto it," Trump said from the Oval Office before officially sending the measure back to Congress without his approval.
House Democrats, meanwhile, were quick to announce they would hold an override vote on March 26. "The House and Senate resoundingly rejected the President's lawless power grab, yet the President has chosen to continue to defy the Constitution, the Congress and the will of the American people," Pelosi said in a statement at the time.
But the House faces a high hurdle, needing a two-thirds majority — rather than a simple majority — to override the veto. The House first passed the bill 245-182, with 13 Republicans voting along with Democrats in favor of overturning the President's national emergency. If all members are present for Tuesday's vote, a successful override would need close to 285 votes — a far cry from 245.
House Democratic leaders are under no illusion that the veto override will pass. Speaker Nancy Pelosi said last week that the point of the vote is to make it clear the new Democratic-controlled House won't tolerate the President's persistence for a border wall.
"Whether we can succeed with the number of votes is not the point," Pelosi said at an event in New York. "We are establishing the intent of Congress."
Senate Minority Leader Chuck Schumer said earlier this month that the National Emergency Act allows for a resolution of disapproval to come up every six months, and Democrats intend to bring it up again then.


Programs Supported by last year's event: Amnesty Club, Anime Club, Awards, Trophies, Plaques, Badminton, Band, Cheer Team, Chess Club, Choir, Cross Country, Cultural Club, CTS Skills and Contests, Dance, Debate Club, Drama Club, Environment Club, Field Hockey, Football, Franco Western Club, Interact Club, Jam Club, Jazz Band, Math Contests, Model UN, Photography Club, Post Secondary Fair, Rugby, Science Club and Contests, Skip and A1 Linkages, Soccer, Swim and Dive, Spectrum, Speech Club, Student Union, Track and Field, Uniforms, Volleyball, Wrestling, Visual Arts, In Town Tournaments, Out of Town Tournaments, RIT, Youth in Action.
Celebrate Western Fundraising Gala, which will be Saturday, April 6, 2019.
Celebrate Western Fundraising Gala is WCHS's one and only annual fundraising event. An evening showcase of impressive student talent - dance, drama, visual art, choral, band and culinary feats, all in one evening, all under one roof.
Listen to the jazz band heat up the night, relax to the harmony of choir, or mingle amongst the impressive array of visual art and multi-media. With delicious food & drink and a classy silent auction, the evening is as impressive as it is important. As a fundraiser, Celebrate Western Fundraising Gala is significant because it benefits ALL students in the school.
Advance tickets are $30 and will be available for sale with school registration in August. Tickets will also be available for sale at the door for $40.
Please consider donating an item for our silent auction, which represents a major portion of our evening's proceeds. Or volunteer! We have an awesome team. So much fun!
Celebrate Western is a private event for the Western Canada High School community.


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Boys Like You Chords
April 7, 2022 Ali Raza 0 Comments
How to Purchase the Boys Like You Chords
ARRANGEMENT DETAILS TAB GIVES YOU DETAILS ABOUT THIS PARTICULAR ARRANGEMENT OF BOYS LIKE YOU
CLICKING ON THE LYRICS TAB GIVES YOU ACCURATE INFORMATION ABOUT THIS PARTICULAR ARRANGEMENT
There are many ways to purchase the Boys Like You chords. You can first click on the tab labeled "ARRANEMENT DETAILS". This will give you all the information you need to buy this particular arrangement. To view the lyrics for this song, click on the LYRICS tab. The following sections will give you the information you need to purchase the Boys Like You chords.
If you're looking for detailed guitar lesson plans, try Make Your Ears Awesome: Chord Progressions. This course consists of 57 lessons that will show you how to play the song's chords. You can sample the first two songs for free by signing up for StringKick All Access membership.
Firstly, the song's key signature is C, which is a relative minor to A. The trend in key signatures is towards fewer flats and sharps. F with one flat is slightly more common than E with three. B has two flats, which makes it the least common key with only 4% of songs using it as a key.
A slash in between two chords indicates an advanced chord, called a polychord. Generally, a polychord consists of two notes played simultaneously. A major chord has three notes while a minor chord only has one. If you're unfamiliar with chords, it's a good idea to take a few notes before moving on to the next section.
The chorus of "Higher" has an interesting and versatile chord progression. This song is often played in drop D tuning, and the chord progression for this song is one of the most popular in rock and pop. The chord progression features an open D and two roots notes at the seventh, and fifth frets. Because of the varying tonal qualities, this tune has a very jazzy feel to it, but is still acoustic friendly.
ARRANGEMENT INFO The ARRANGEMENT INFO TABLE GIVES A DESCRIPTION OF THIS PARTicular arrangement of boys like you chords. The tab shows the alphabetical order of the notes. The notes of the A, B, and D are in ascending order.
A RHYTHM: Western and rock songs use a four beat backbeat. The drummer on the snare drum often performs accents. The rhythm section plays the chord-playing and bass instruments, such an electric bass. A double bass is also used in jazz music. This section also includes drums.
ARRANGEMENT INFO Tab INCLUDES ALL ARRANGEMENT DETAILS
A NOTE FOR NOTE SOLO – Another example
You can find the correct lyrics online if you are unsure of the name of a song or chord. A few clicks will give you accurate information about the song. You can simplify the chords and change their colors. By hovering your mouse over the chords, you can find the name of the chord.
You can adjust the meter on your hookpad to suit your song. This setting lets you choose whether the melody is in scale or out of it. The hookpad also features a loop cycle bar, which you can enable or disable, so that you can hear the song over again.
You can edit the notes of the song in the final step. You can adjust the spacing between notes, and the notes will be automatically transposed. The default setting for music is "table mode". You can also enter a single note in the music with a rest in the space between. This will preserve the rhythmic spacing.
If you want to see the lyrics of a particular song, click on the lyric tab. It provides accurate information about the song. Each chord has its own note symbol on the tab. The chord symbol can be placed below the fretboard diagram. It will automatically align all elements according to system and current measure.
A major chord is defined as the next chord below the root of the first. If a minor chord is used to backdoor, it will become an I chord. In a major key, this will be C major. A minor chord would be F major. A song in this mode will have a j or k chord in the second position.
Another useful tool for learning to play songs on the ukulele is a chord chart. This feature displays the chords and strum patterns for the song. The lyrics are also displayed above these chords. This is very useful for beginners, as they may not be able to distinguish between a tab or the lyrics.
A song's key signature will help you determine whether it is a polyphonic or a monophonic arrangement. Monophonic music uses one rhythm for each section, while polyphonic music uses two melodies that do not belong to the same key. Polyphonic pieces are usually in a minor key. The first part is played in one key and the second part in another.
Besides a chord chart, a lead sheet displays the melody line. Chord charts are easier to read than lead sheets. While lead sheets are used in pop and jazz songs, classical pieces are usually notated using the treble clef. Guitarists should know both notation systems to pick up new songs quickly. Besides, reading guitar tabs will help you explore various finger picking patterns.
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Christopher Surfleet GIA AJP
Auctioneer & Senior Valuer, The Cheshire Saleroom
chris@adampartridge.co.uk
Chris started his career in 1992 with Phillips Auctioneers, working his way up to the position of valuer. He then joined Bonhams as a Regional Valuer, before working in the picture department. He began at Adam Partridge Auctioneers & Valuers in 2011 and specialises in a wide range of areas including paintings, watches, works of art and jewellery, qualifying with the Gemological Institute of America in 2015. His interests also spread to early English glass, Japanese ceramics and classic cars. He has been responsible for presiding over some of the largest collections Partridge's have sold, including the Reginald Cheetham collection of militaria and scientific instruments, The Roger Haynes collection of works of art, The Jam Rock & Pop auction, The Exhibition & Exposition auction, The Sean Rice collection and the James Northcote Nash Collection of Art. Chris has also been responsible for cataloguing several of the on-site auctions held including the contents of the Vina Cooke Doll Museum, Summerfield House and for Liberty & Co. Chris now splits his time between The Cheshire Saleroom and his home in Devon, where he is setting up a South West satellite office.
Areas of Specialism:
Silver, Jewellery & Watches, Paintings, Sporting, Studio Ceramics & Modern Design, Ceramics, Furniture & Clocks, Asian Art, Stamps & Ephemera, Bronzes & Sculpture


MGM Bridgeport is a Casino Room in Connecticut. It has 2000 slot machines, 160 table games.
Gargantuan resort-hotels may populate the skyline in Las Vegas, a city that resides in what some call a "cosmopolitan sophistication", but for the leisurely gambler in Connecticut, location and price are the most important variables that figure into his or her decision on where to have a good time.


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Home » Celebrities » Janet Jackson references #MeToo in Billboard speech
Janet Jackson references #MeToo in Billboard speech
Superstar and Billboard Icon Award recipient Janet Jackson made her stance on the MeToo movement clear during her acceptance speech at the awards show Sunday night, and thanked God for her artistic career.
"I believe that, for all of our challenges, we live at a glorious moment in history," she began, after performing her 1986 hit "Nasty" and 1993 song "Throb." "It's a moment that, at long last, women have made it clear that we will no longer be controlled, manipulated, or abused. I stand with those women and with those men equally outraged by discrimination, who support us in heart and mind."
She continued with a message about faith. "My prayer is that, weary of such noise, we will turn back to the source of all calmness, that source is God. Everything we lack, God has in abundance: compassion, sensitivity, patience and boundless love. Again I want to thank all of you for this honor and I thank God for giving me the precious energy that lets me live my life as an artist who every day seeks to expend my capacity to love."
Jackson is the first black woman to receive the Icon Award, as surprise presenter Bruno Mars pointed out in his introduction.
"The name 'Jackson' represents artistic genius and iconic performance," said Mars of the legendary superstar. "The Jacksons are music royalty and the first family of entertainment. She is an activist. She's a humanitarian. She's a powerful woman."
« Samantha Markle accuses Meghan's mother Doria Ragland of 'cashing in'
Billboard Awards 2018 winners list in full sees Ed Sheeran, Taylor Swift and BTS take home gongs »


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IL-21 Up-Regulates the Expression of Genes Associated with Innate Immunity and Th1 Response
Mari Strengell, Timo Sareneva, Don Foster, Ilkka Julkunen and Sampsa Matikainen
J Immunol October 1, 2002, 169 (7) 3600-3605; DOI: https://doi.org/10.4049/jimmunol.169.7.3600
Mari Strengell
Department of Microbiology, National Public Health Institute, Helsinki, Finland; and
Timo Sareneva
Don Foster
Department of Functional Cloning, ZymoGenetics, Seattle, WA 98102
Ilkka Julkunen
Sampsa Matikainen
IL-21 is a recently characterized T cell-derived cytokine that regulates NK and T cell function. IL-21R shares the common γ-chain (γc) with the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Despite the same γc, these cytokines have different effects on diverse cells. In this study, we have studied IL-15- and IL-21-induced gene expression in human primary NK and T cells and the NK-92 cell line. Both IL-15 and IL-21 rapidly induced mRNA synthesis for IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and myeloid differentiation factor 88 (MyD88), the genes that are important in activating innate immunity and Th1 response. IL-15 induced STAT5 DNA binding to the IL-2Rα IFN-γ-activated sequence (GAS), MyD88 GAS, and c-sis-inducible elements, whereas IL-21 induced STAT3 DNA binding to MyD88 GAS and c-sis-inducible elements. IL-21-induced STAT3 activation was verified by immunoprecipitation and Western blotting with anti-phosphotyrosine Ab. In addition, pretreatment of NK-92 cells with IL-15 or IL-21 strongly enhanced IL-12-induced STAT4 DNA binding to IL-2Rα GAS. The induction of IFN-γ, T-bet, IL-12Rβ2, and IL-18R gene expression in NK cells, along with STAT3 activation, suggests that IL-21 is involved in the activation of innate immune responses. Moreover, the enhanced transcription of these genes in T cells establishes a significant role for IL-21 also in the Th1 response.
Cytokines are a large and diverse group of molecules that mediate proliferation, differentiation, and survival in hemopoietic cells. Despite the heterogeneity of the members of the cytokine family, many of them have overlapping effects. This can be partly explained by the fact that the receptors for different cytokines often share the same receptor subunit. Recently, a novel cytokine, IL-21, was characterized (1). IL-21 is a T cell-derived cytokine that has important functions in NK and T cells. It enhances anti-CD3-induced proliferation of naive human T cells and activates STAT3 in the human T cell leukemia virus I-transformed human T cell line (1, 2). IL-21R, expressed on both NK and T cells (1), is related to IL-2Rβ (3). It was recently shown that the common γ-chain (γc),3 shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is also the functional component of the IL-21R complex (2). Mice lacking the IL-2/IL-15Rβ chain do not have NK and T cells. On the other hand, NK and T cell development is normal in mice lacking the IL-2 gene, suggesting that other cytokines using IL-2/IL-15Rβ are important for the development of NK and T cells (4).
NK cells have a crucial role in innate immunity, especially against viral infections and in the control of tumor growth. NK cells exhibit cytotoxic activities toward cells with diminished expression of MHC class I molecules on their surface (5). Macrophage-derived cytokines IFN-α, IL-12, and IL-18 enhance NK cell cytotoxicity and activate NK cells to produce IFN-γ and TNF-α (6, 7, 8, 9, 10). NK cell-produced IFN-γ is involved in the polarization of Th cells toward the Th1 type. IL-12 is also required for the development of the Th1-type immune response (11) and it enhances IFN-γ production in NK and T cells, which further stimulates the capacity of macrophages and dendritic cells to carry out Ag presentation.
In the present work, we have studied IL-15- and IL-21-induced gene expression and signaling in human NK and T cells. We show that both IL-15 and IL-21 induce mRNA expression of IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and myeloid differentiation factor 88 (MyD88) genes that are involved in innate immunity and the Th1 response. We also demonstrate that IL-21 preferentially activates STAT3, whereas IL-15 activates STAT5 both in NK and T cells. In addition, we show that priming of NK-92 cells with IL-15 or IL-21 enhances IL-12-induced STAT4 DNA binding, further suggesting that IL-15 and IL-21 promote the Th1 response.
Primary T cell culture
Leukocyte-rich buffy coats were obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). Mononuclear cells were isolated from buffy coats by density gradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). Monocytes were removed by adherence on plastic plates and nonadherent T cells were further purified through a nylon wool column. Purified T cells were activated with 0.5 μg/ml anti-CD3 and 0.5 μg/ml anti-CD28 mAbs (R&D Systems, Abingdon, U.K.) and cultured in RPMI 1640 medium supplemented with 10% FCS (Integro, Zaandam, The Netherlands), 20 mM HEPES, 2 mM l-glutamine, 0.6 μg/ml penicillin, 60 μg/ml streptomycin, and 100 IU/ml human rIL-2 (R&D Systems) for 5–6 days. T cells were then propagated for 5–6 days in RPMI 1640 supplemented with 100 IU/ml IL-2. In each experiment, T cells from two to four donors were used.
Enrichment of primary NK cells
Mononuclear cells were isolated by density gradient centrifugation as described above using Ficoll-Paque. NK cells were purified from nonadherent PBMCs through a nylon wool column and two-step density gradient centrifugation by Percoll (Amersham Pharmacia Biotech), and then followed by purification with immunomagnetic beads coated with anti-CD3, anti-CD14, and anti-CD19 Abs (Dynal, Oslo, Norway). As determined by flow cytometry with anti-CD16 or anti-CD56 Abs, NK cells were >90% pure.
NK cell line
A human NK-92 cell line (CRL-2407; American Type Culture Collection, Manassas, VA) was maintained in MEM α medium (Life Technologies, Carlsbad, CA) supplemented with 12% horse serum (Life Technologies), 12% FCS, 0.2 mM i-inositol, 20 mM folic acid, 40 mM 2-ME, 2 mM l-glutamine, antibiotics, and 100 IU/ml IL-2.
Cell stimulation
Human rIL-21 (ZymoGenetics, Seattle, WA) was used at 10 ng/ml. Human rIL-15 (R&D Systems) was used at 5 ng/ml. Despite the fact that the cells presumably consumed all IL-2 from the media during propagation, the cells were cultured in IL-2-free RPMI 1640 for 1 h (primary T cells) or for 18 h (NK-92 cell line) before stimulation with IL-15 or IL-21.
RNA isolation and Northern blot analysis
Total cellular RNA was isolated as previously described (12). Total cellular RNA was quantified photometrically and the samples containing equal amounts of RNA (10 μg) were size fractionated on a 1.0% formaldehyde-agarose gel and transferred to a nylon membrane (Hybond; Amersham, Buckinghamshire, U. K.). The membrane was hybridized with the cDNA probes for human IFN-γ (13), T-bet, IL-2Rα, IL-12Rβ2, IL-18Rα (14), IL-18Rβ (15), and MyD88 (16). T-bet and IL-12Rβ2 probes were cloned from total cellular RNA obtained from IL-15-, IL-18-, and IL-21-treated NK-92 cells by RT-PCR using oligonucleotides CCGCCTGGATCCAACTGTCAATTC (sense), ATCATGGGATCCGCTCAGTTGGGA (antisense), AGGGAAAAAGGATCCCAAGGTCAT (sense), and AGACCAACTCCCGGATCCTAAGAC (antisense), respectively. Ethidium bromide staining of ribosomal RNA bands was used to ensure equal RNA loading. The probes were labeled with [α-32P]dCTP (3000 Ci/mmol; Amersham) using a random primed DNA labeling kit (Boehringer Mannheim, Mannheim, Germany). The membranes were hybridized (Ultrahyb; Ambion, Austin, TX) and washed twice at 42°C and once at 60°C in 1× SSC/0.1% SDS for 30 min each time, and exposed to Kodak AR X-Omat films at −70°C using intensifying screens.
Nuclear extracts and nuclear protein/DNA-binding reactions were performed as described previously (17, 18). IL-2Rα IFN-γ-activated sequence (GAS), GAS-c/GAS-n, 5′-GATCTTTCTTCTAGGAAGTACCAAACATTTCTGATAATAGAA-3′; MyD88 GAS, 5′-GATCGGAGCTTCTCGGAAAGCGAAAGAAGGA-3′; and c-sis-inducible element (SIE), 5′-GATCGATCTAGGGATTTCCGGGAAATGAAGCT-3′ oligonucleotides were purchased from DNA Technology (Aarhus, Denmark). The probes were labeled with [32P]dATP by using Klenow polymerase. The protein/DNA-binding reaction was done at room temperature for 30 min and the samples were analyzed by electrophoresis on 6% nondenaturing low-ionic strength polyacrylamide gels in 0.25× Tris-borate-EDTA buffer. The gels were dried and visualized by autoradiography. Anti-STAT1 (sc-345X), anti-STAT3 (sc-482X), anti-STAT4 (sc-486X), and anti-STAT5b (sc-835X; recognizes both STAT5a and STAT5b) Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Antiphosphotyrosine analysis
Cytokine-stimulated cells were lysed in immunoprecipitation binding buffer (50 mM Tris (pH 7.4), containing 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100) supplemented with protease inhibitors (Complete; Boehringer Mannheim) and 1 mM sodium orthovanadate. Cell lysates were immunoprecipitated on ice for 1 h with anti-Janus kinase (JAK) 1 (sc-295, Santa Cruz Biotechnology), anti-JAK3 (sc-513, Santa Cruz Biotechnology), anti-STAT3, or anti-STAT5b Abs. The proteins were separated on 8 or 10% SDS-PAGE, transferred onto Immobilon-P membranes (Millipore, Bedford, MA), and analyzed by anti-phosphotyrosine Ab (PY99; Santa Cruz Biotechnology) as described previously (12, 14).
IL-21 induces the expression of genes associated with activation of innate immunity and Th1 response
IL-15 and IL-21 are important NK and T cell regulatory cytokines playing a significant role in their activation and proliferation. We analyzed the effect of these cytokines on the expression of IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18Rα, IL-18Rβ, and MyD88 genes. NK-92 and T cells were stimulated with IL-15 or IL-21, harvested, and the total cellular RNA was isolated. Both IL-15 and IL-21 induced the expression of the IFN-γ gene in NK-92 cells (Fig. 1⇓) and T cells (Fig. 2⇓). In NK-92 cells, IL-15-induced steady-state IFN-γ mRNA levels were maximum 3 h after stimulation, whereas in T cells the expression peaked at 1 h after stimulation. T-bet is a recently characterized transcription factor that regulates the activation of innate immunity and polarization of T lymphocytes toward Th1-type cells (19). Both IL-15 and IL-21 induced T-bet gene expression in NK-92 cells. The up-regulation of the T-bet gene by IL-15 was rapid, reaching a maximum at 1–3 h after stimulation, whereas the kinetics of IL-21-induced T-bet mRNA expression was somewhat slower. In T cells, IL-21 was a more potent up-regulator of T-bet mRNA levels compared with that of IL-15.
Kinetics of IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and MyD88 mRNA expression in NK-92 cells stimulated with IL-15 or IL-21. Cells were stimulated with IL-15 (5 ng/ml) or IL-21 (10 ng/ml) for the times indicated and collected, and the total cellular RNA was isolated and prepared for Northern blot analysis with IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, and MyD88 probes. Ethidium bromide staining of rRNA bands was used to ensure equal RNA loading. The results are representative of three independent experiments.
Kinetics of IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and MyD88 mRNA expression in T cells stimulated with IL-15 or IL-21. T cells from four individual blood donors were stimulated separately with IL-15 or IL-21 for the indicated times. The cells were collected and pooled, and total cellular RNA was isolated and prepared for Northern blot analysis with IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, and MyD88 probes. The results shown are representative of three separate experiments.
Both IL-15 and IL-21 up-regulated the expression of the IL-2Rα gene in NK-92 cells (Fig. 1⇑). In T cells, IL-15 enhanced the expression of the IL-2Rα gene more efficiently compared with that of IL-21, whereas in NK-92 cells both cytokines induced IL-2Rα mRNA synthesis equally well (Figs. 1⇑ and 2⇑). Both IL-15 and IL-21 up-regulated IL-12Rβ2 expression in NK-92 cells, as has previously been shown in T cells (20). In T cells, IL-15-induced IL-12Rβ2 mRNA expression was more efficient compared with that of IL-21. (Figs. 1⇑ and 2⇑). As shown in Figs. 1⇑ and 2⇑, IL-15 and IL-21 up-regulated the expression of IL-18Rα and IL-18Rβ in NK-92 and T cells.
IL-15 induces STAT5 DNA binding to the IL-2Rα GAS-c/GAS-n element in NK-92 cells
The IL-2Rα gene contains an upstream STAT5-binding element that is essential for IL-2-induced IL-2Rα transcription (21, 22). To characterize IL-15- and IL-21-induced STAT DNA binding to the GAS-c/GAS-n element (from −3778 to −3740) of the IL-2Rα gene, NK-92 cells were stimulated with IL-15 or IL-21 for different time periods, and nuclear extracts were prepared followed by analysis with EMSA. As shown in Fig. 3⇓A, EMSA with the IL-2Rα GAS-c/GAS-n oligonucleotide revealed the induction of one major protein-DNA complex in response to IL-15 stimulation. The IL-15-induced complex was already seen at 1 h after the cytokine stimulation. The complex induced by IL-15 was identified with specific anti-STAT Abs. Both anti-STAT1 and anti-STAT5 Abs supershifted the IL-15-induced complex (Fig. 3⇓B). IL-21 stimulation of NK-92 cells did not induce detectable STAT5 binding to IL-2Rα GAS-c/GAS-n.
IL-15 induces STAT5 binding to the IL-2Rα promoter GAS element. A, NK-92 cells were stimulated for the indicated times with IL-15 (5 ng/ml) or IL-21 (10 ng/ml), and nuclear extracts were prepared. The extracts were incubated with the IL-2Rα GAS probe and analyzed by EMSA. B, The nuclear extracts from IL-15-induced cells were pretreated with anti-STAT1, anti-STAT3, anti-STAT4, or anti-STAT5 Abs for 1 h followed by EMSA with the IL-2Rα probe. Comparable data were obtained from three independent experiments.
IL-21 induces STAT3 binding to MyD88 GAS and SIE in NK-92 cells
In addition to the IL-2Rα gene, both IL-15 and IL-21 up-regulated MyD88 gene expression. MyD88 is a cytoplasmic adapter protein involved in IL-18 and Toll-like receptor signal transduction and it is an essential factor in innate immunity (23, 24). To study possible STAT binding to MyD88 GAS, we stimulated NK-92 cells with IL-15 or IL-21, prepared nuclear extracts, and analyzed them by EMSA. As shown in Fig. 4⇓A, both IL-15 and IL-21 induced STAT binding to MyD88 GAS. The differential mobility of IL-21- and IL-15-induced protein-DNA complexes in the gel suggested that these cytokines activate different STATs. To identify IL-15- and IL-21-induced complexes, we performed supershift experiments with anti-STAT Abs. Anti-STAT5 and anti-STAT1 Abs supershifted both of the IL-15-induced MyD88 GAS complexes, whereas anti-STAT3 Ab supershifted IL-21-induced MyD88 GAS binding complexes (data not shown).
IL-21 induces STAT3 binding to MyD88 GAS and SIE. NK-92 cells were stimulated with IL-15 or IL-21 for the indicated times and nuclear extracts were prepared and analyzed by EMSA with MyD88 GAS (A) and SIE probes (B). C, Nuclear protein extracts were incubated with anti-STAT Abs for 1 h, followed by binding to SIE. The experiments were repeated three times with similar results.
SIE is a well-characterized element binding activated STAT3 (25). Both IL-15 and IL-21 induced strong STAT binding to the SIE element (Fig. 4⇑B). The cytokine-induced protein-DNA complexes were identified by supershift assays. IL-15-induced complexes were supershifted at least partially with anti-STAT1, anti-STAT3, and anti-STAT5 Abs (Fig. 4⇑C). In contrast, the IL-21-induced complex was completely supershifted with anti-STAT3 Ab (Fig. 4⇑C).
IL-21 induces STAT3 binding in primary NK and T cells
Next, we analyzed whether IL-21 was able to activate STAT3 also in primary human NK and T cells. NK and T cells were purified as described in Materials and Methods. The cells were stimulated with IL-15 or IL-21 and nuclear extracts were prepared followed by EMSA. Similar to NK-92 cells, IL-15 induced STAT5 and IL-21 induced STAT3 DNA binding to SIE in primary NK cells (Fig. 5⇓A). Anti-STAT5 Ab supershifted the IL-15-induced protein-DNA complex, whereas the IL-21-induced complex was supershifted by anti-STAT3 Ab. In contrast to NK cells, stimulation of primary T cells with IL-21 induced both STAT3 and STAT5 binding to SIE (Fig. 5⇓B).
IL-21 induces STAT3 binding to SIE in primary NK and T cells. Human primary NK cells (A) or T cells (B) from four individual donors were stimulated with IL-15 or IL-21. After a 1-h stimulation, the cells were collected, and nuclear extracts were prepared and incubated with anti-STAT1, anti-STAT3, anti-STAT4, or anti-STAT5 Abs, followed by EMSA with a SIE probe.
IL-21 activates STAT3 tyrosine phosphorylation
Since IL-21 induced STAT3 DNA binding to SIE and MyD88 GAS probes, we verified STAT3 activation with antiphosphotyrosine analysis. NK-92 and T cells were stimulated with IL-15 or IL-21 and proteins in cytokine-stimulated cell lysates were immunoprecipitated with anti-STAT3 or anti-STAT5 Abs followed by Western blot analysis with anti-phosphotyrosine Ab. As a control, cells were stimulated with IFN-α which is known to activate multiple STAT proteins (13). In unstimulated NK-92 cells, STAT3 or STAT5 tyrosine phosphorylation was not detectable, whereas IFN-α, IL-15, and especially IL-21 induced very efficiently STAT3 tyrosine phosphorylation (Fig. 6⇓A). Very strong STAT5 tyrosine phosphorylation was seen in IL-15-stimulated NK-92 cells. In addition, a weak tyrosine phosphorylation of STAT5 was also detected in cells stimulated with IFN-α or IL-21 (Fig. 6⇓A). Similar to NK-92 cells, IFN-α and IL-21 induced STAT3 tyrosine phosphorylation in T cells (Fig. 6⇓B), whereas IL-15 stimulation of T cells activated STAT5 tyrosine phosphorylation (Fig. 6⇓B).
Induction of tyrosine phosphorylation of STAT3 and STAT5 by IFN-α, IL-15, and IL-21 in NK-92 and T cells. NK-92 cells (A) or T cells (B) were stimulated with IFN-α, IL-15, or IL-21. The cells were collected and cell lysates were prepared and immunoprecipitated with anti-STAT3 or anti-STAT5 Abs. Immunoprecipitated proteins were separated on 10% SDS-PAGE, transferred to membranes, and stained with an anti-phosphotyrosine (P-Y) Ab. The membranes were stripped and restained with anti-STAT3 and anti-STAT5 Abs to ensure equal sample loading. A representative of three separate experiments is shown.
IL-21 induces JAK1 and JAK3 tyrosine phosphorylation
A recent study by Asao et al. (2) suggested that IL-21 stimulation lead to the activation of JAK1 and JAK3 in a γc-transfected T cell line. We analyzed whether JAK1 or JAK3 was activated by IL-21 also in NK and T cells. For that, NK-92 and T cells were stimulated with IL-15 or IL-21 and proteins in cell lysates were immunoprecipitated with anti-JAK1 or anti-JAK3 Abs followed by Western blot analysis with an anti-phosphotyrosine Ab. IL-15 and IL-21 induced JAK3 tyrosine phosphorylation in both cell types efficiently (Fig. 7⇓). Instead, IL-15 and IL-21 had a weak enhancing effect on JAK1 tyrosine phosphorylation (Fig. 7⇓).
IL-21 induces tyrosine phosphorylation of JAK1 and JAK3 in NK-92 and T cells. NK-92 (left panel) and T cells (right panel) were stimulated with IL-15 or IL-21. After a 30-min stimulation, the cells were collected and lysed, and the cell lysates were immunoprecipitated with anti-JAK1 or anti-JAK3 Abs. The immunoprecipitated proteins were separated on 8% SDS-PAGE, transferred to membranes, and blotted with an anti-phosphotyrosine (P-Y) Ab. To ensure equal protein loading, the membranes were stripped and restained with anti-JAK1 and anti-JAK3 Abs. The results shown are representative of two independent experiments.
IL-15 and IL-21 priming enhances IL-12-induced STAT4 activation
IL-12 is a key cytokine in regulating NK and T cell activation (26). Because IL-15 and IL-21 stimulation of NK-92 cells enhanced IL-12Rβ2 gene expression, we studied whether pretreatment of cells with IL-15 or IL-21 would result in enhanced IL-12-induced STAT4 DNA binding. For that, NK-92 cells were pretreated with IL-15 or IL-21 for 18 h and after that the cells were left untreated or stimulated with IL-12 for 1 h followed by EMSA with IL-2Rα GAS-c/GAS-n probe. Both IL-15 and IL-21 priming enhanced IL-12-stimulated STAT4 binding to IL-2Rα GAS-c/GAS-n (Fig. 8⇓).
Pretreatment of NK-92 cells with IL-15 or IL-21 enhances IL-12-induced STAT4 DNA binding. NK-92 cells were incubated for 18 h with IL-15 (5 ng/ml) or IL-21 (10 ng/ml). The cells were washed and left untreated or stimulated with 1 or 5 ng/ml IL-12 for 1 h. Cells were collected, and nuclear extracts were prepared and analyzed by EMSA with the IL-2Rα GAS probe. The experiment was repeated twice with similar results.
Previous studies have shown that IL-21 has an important role in NK, B, and T cell functions (1). IL-21 is structurally related to IL-2 and IL-15, the cytokines that have a crucial role in thymic and peripheral T cell maturation, respectively (27, 28). IL-15 regulates NK and CD8+ memory cell maturation and acquisition of cytolytic activity. Studies with IL-15 and IL-15R KO mice have shown that the lack of IL-15 or IL-15Rα leads to a severe reduction or absence of NK cells (29, 30). In contrast, constitutive production of IL-15 over several months in IL-15-transgenic mice led to the development of T-NK lymphocytic leukemia (31). In the present study, we investigated the effect of IL-15 and IL-21 on the expression of genes that are important for NK and T cell function. In this report, we show that IL-15 and IL-21 stimulation of human NK and T cells induces the expression of IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and MyD88 genes.
IFN-γ has a major role as a regulator of innate and adaptive immune responses. IFN-γ promotes innate and Th1 immune responses by activating macrophages, NK cells and T cells, as well as by enhancing Ag presentation (32). The results reported here show that IL-15 and IL-21 enhance the expression of the IFN-γ gene in NK and T cells. Both IL-15 and IL-21 up-regulated IFN-γ mRNA synthesis already at 1 h after stimulation, suggesting that these cytokines directly activate IFN-γ gene expression at the transcriptional level. IFN-γ production is induced by TCR ligation or macrophage-derived cytokines such as IFN-α, IL-12, and IL-18 (33, 34, 35). IL-18 is essential for IFN-γ production both in bacterial and viral infections (36, 37). IL-18R consists of two subunits, IL-18Rα and IL-18Rβ (37). IL-18Rα belongs to the IL-1R family, using the IL-1R-associated kinase-NF-κB signaling pathway. Activation of these receptors requires an adapter protein, MyD88 (38, 39). We demonstrate that IL-15 and IL-21 enhance mRNA expression of IL-18R and MyD88. Moreover, our results suggest that these cytokines also indirectly enhance IFN-γ production and, consequently, Th1 response by inducing IL-18R expression in NK cells.
IL-12 is the major cytokine driving Th1 differentiation. Th1 differentiation also requires transcription factor T-bet that activates IFN-γ and IL-12Rβ2 gene expression (19). A recent study shows that T-bet is required for IFN-γ production in CD4 and NK cells, but not in CD8 T cells (40). T-bet can also stimulate indirectly its own gene expression by inducing IFN-γ, which in turn activates T-bet gene expression (41, 42). We demonstrate here that both IL-15 and IL-21 up-regulate the mRNA synthesis for T-bet and IL-12Rβ2 genes in NK and T cells. Furthermore, pretreatment of NK-92 cells with IL-15 or IL-21 resulted in enhanced IL-12-induced STAT4 DNA binding. These results suggest that both IL-15 and IL-21 have effects that can enhance a Th1-type immune response.
Receptors for IL-2, IL-15, and IL-21 share the same γc (2, 43). In addition, the IL-2/IL-15Rβ subunit that is essential for the proliferation of NK and T cells is also closely related to IL-21R. IL-2 and IL-15 utilize STAT5 in their intracellular signal transduction and STAT5 is essential for IL-2-induced T cell proliferation (44). Asao et al. (2) demonstrated that IL-21 uses γc in signaling and activates STAT3 in a human T cell leukemia virus I-transformed human T cell line. They also showed that the phosphorylation of JAK1 and JAK3 was enhanced by IL-21. We show here that in primary T cells and NK-92 cells IL-21- and especially IL-15-induced JAK3 tyrosine phosphorylation was efficient. In contrast, IL-21 had a modest enhancing effect on JAK1 tyrosine phosphorylation in both cell types.
As shown in the present report, IL-15 stimulation of NK-92 cells activated STAT5 DNA binding to the IL-2Rα promoter element and induced IL-2Rα mRNA synthesis. Similar IL-15-induced STAT5 DNA binding was also detected in human primary NK and T cells. The results are consistent with an earlier report demonstrating that STAT5 and, to some extent, STAT1 and STAT3 are activated by IL-15 in NK and T cells (45). Using MyD88 GAS and SIE elements in EMSA and analysis of STAT tyrosine phosphorylation, we demonstrated that IL-21 activates STAT3 not only in human NK-92 cell line but also in human primary NK and T cells. In NK-92 cells, IL-21-induced STAT binding to the IL-2Rα promoter element was not detected although the mRNA synthesis for IL-2Rα was enhanced. Similarly, IL-21 was not able to induce STAT DNA binding to recently described IL-2Rα GAS elements residing in the first intron of the IL-2Rα gene (data not shown) (46). These results suggest that IL-21 activates other signaling pathways, leading to the activation of the IL-2Rα gene. In conclusion, our results demonstrate that IL-15 and IL-21 enhance the innate as well as Th1 immune response by activating different STATs, which is followed by the induction of IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and MyD88 gene expression in NK and T cells.
We thank Dr. John E. Sims (Immunex, Seattle, WA) for providing IL-18Rα and MyD88 cDNAs and Teija Westerlund and Marika Yliselä for expert technical assistance.
↵1 This work was supported by the Medical Research Council of the Academy of Finland, the Sigrid Juselius Foundation, and the Finnish Cancer Foundations.
↵2 Address correspondence and reprint requests to Dr. Mari Strengell, Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail address: mari.strengell{at}ktl.fi
↵3 Abbreviations used in this paper: γc, common γ-chain; GAS, IFN-γ-activated sequence; SIE, c-sis-inducible element; JAK, Janus kinase; MyD88, myeloid differentiation factor 88.
Accepted July 24, 2002.
Copyright © 2002 by The American Association of Immunologists
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Mari Strengell, Timo Sareneva, Don Foster, Ilkka Julkunen, Sampsa Matikainen
The Journal of Immunology October 1, 2002, 169 (7) 3600-3605; DOI: 10.4049/jimmunol.169.7.3600
Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
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Shopblocks secures £300k investment from Northern angel network
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