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2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN- Γ Release in Human NK Cells Involves Distinct Pathways

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2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN- Γ Release in Human NK Cells Involves Distinct Pathways 2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN- γ Release in Human NK Cells Involves Distinct Pathways This information is current as Samuel S. Chuang, Pappanaicken R. Kumaresan and of October 2, 2021. Porunelloor A. Mathew J Immunol 2001; 167:6210-6216; ; doi: 10.4049/jimmunol.167.11.6210 http://www.jimmunol.org/content/167/11/6210 Downloaded from References This article cites 79 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/167/11/6210.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN-␥ Release in Human NK Cells Involves Distinct Pathways1 Samuel S. Chuang, Pappanaicken R. Kumaresan, and Porunelloor A. Mathew2 2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-␥ and matrix metal- loproteinases, and NK cell invasiveness. Although there has been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various specific inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-␥ release indicating distinct Downloaded from pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways. The Journal of Immunology, 2001, 167: 6210–6216. http://www.jimmunol.org/ atural killer cells recognize and kill virally infected cells, (SHP)3 1 and/or SHP-2. These phosphatases can dephosphorylate parasites and certain tumor cells (1–3). NK cells play an several substrates within the activating signaling cascade or a dis- N important role by their ability to target MHC class I tal activating receptor and inhibit NK cell activation (14, 15). negative cells that escape recognition by cytotoxic T cells (4, 5). In Although there has been great progress in understanding the addition to cytolytic activity, NK cells also produce several regu- inhibitory mechanisms that regulate NK cell function, our knowl- latory cytokines including IFN-␥, TGF␤, IL-1␤, IL-10 and GM- edge of the activating signaling pathways is slowly emerging. Sev- CSF as well as matrix metalloproteinases (MMPs) (6–9). The eral surface molecules have been identified that can activate nat- mechanisms that control NK cell activation and cytotoxicity are ural cytotoxicity. Activating receptors such as 2B4, CD2, CD16, by guest on October 2, 2021 believed to be determined by a delicate balance between stimula- KIR3DS1-5, NKp30, NKp44, and NKp46 are members of the Ig tory and inhibitory signals received from surface receptors (10– superfamily (16–18). These molecules bear high homology on 12). NK cells also mediate the rejection of MHC mismatched bone their extracellular domains while lacking the immunoreceptor ty- marrow stem cells (13). Engagement of cytolytic activity can be rosine-based inhibitory motifs on their cytoplasmic tails. There are inhibited by MHC class I molecules on target cells interacting with also members of the lectin gene superfamily that can transduce an MHC class I receptors expressed on the surface of NK cells. NK activating signal. Members of the lectin superfamily, such as cell inhibitory receptors belong to the Ig and lectin gene super- NKR-P1A and NKR-P1C, NKG2 family, and the LY49 family can families. One common feature of the killer cell inhibitory receptors form homo- and heterodimer complexes that can be inhibitory or is the presence of immunoreceptor tyrosine-based inhibitory mo- stimulatory. Although many of the activating surface molecules tifs (ITIMs) in their cytoplasmic domains (3, 10). The ITIM motifs have been identified, information on the signaling cascade from the become phosphorylated upon receptor binding which results in the cell surface to within is fragmentary. recruitment of protein SH2 domain bearing tyrosine phosphatase 2B4 was originally identified on mouse NK cells and the subset of T cells that mediate non-MHC-restricted killing (18–21). It is a novel member of the CD2 subfamily which includes signaling lymphocyte-activation molecule, CD48, CD58, CD84, CS1, and Department of Molecular Biology and Immunology and Institute for Cancer Re- Ly9, and is expressed on all NK cells, a subset of CD8ϩ T cells, search, University of North Texas Health Science Center, Fort Worth, TX 76107 basophils, and monophils (22–24). The cytoplasmic domain of Received for publication March 28, 2001. Accepted for publication September 26, 2001. 2B4 contains novel tyrosine motifs (TxYxxV/I) that associate with signaling adaptor molecule, signaling lymphocyte-activation mol- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ecule-associated protein (SAP) whose defect forms the basis for with 18 U.S.C. Section 1734 solely to indicate this fact. X-linked lymphoproliferative syndrome (XLP) (25–28). It is 1 This work was supported in part by National Institutes of Health Grants AI 38938 and CA 85753. This work was supported in part by a grant from the University of North Texas Health Science Center Institute for Cancer Research. We appreciate 3 Abbreviations used in this paper: SHP, SH2 domain bearing tyrosine phosphatase; support of our work from Bank One through the University of North Texas Health SAP, signaling lympocyte-activation molecule-associated protein; XLP, X-linked Science Center Institute for Cancer Research. lymphoproliferative syndrome; MAPK, mitogen-activated protein kinase; ADCC, 2 Address correspondence and reprint requests to Dr. Porunelloor A. Mathew, De- Ab-dependent cellular cytotoxicity; ERK, extracellular signal-regulated kinase; LAT, partment of Molecular Biology and Immunology, University of North Texas Health linker for activation of T cells; h2B4, human 2B4; MEK1, MAPK kinase 1; rADCC, Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699. E-mail redirected ADCC; GEM, glycolipid-enriched microdomains; MMP, metalloprotein- address: [email protected] ase; PLC, phospholipase C. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 6211 thought that NK cells in XLP patients are defective in their acti- 51Cr release cytotoxicity assay vation through 2B4 signaling (29–31). In addition to modulating K562 cells and P815 cells, where indicated, were used as target cells and cytolytic function, 2B4 activation of NK cells induces cytokine ϫ 6 51 labeled by incubating 1 10 cells with 2 MBq of Na2 CrO4 (NEN production as well as invasiveness (9, 19, 32). CD48 was recently Research Products, Boston, MA) for 90 min at 37°C under 5% CO2 in air. identified as the high-affinity counterreceptor of 2B4 in both mice The target cells were then washed three times in culture medium. Ten ␮ and humans (33, 34). CD48–2B4 interactions are physiologically thousand labeled target cells (100 l) were incubated with the effector YT cell suspension (100 ␮l), with and without mAb C1.7 (200 ng/ml). Effector important since they enhance the lytic function of human NK cells YT cells were resuspended and added at 1, 2, 5, 10, and 20 times the (23). It has been reported that 2B4 may function as a coreceptor in number of labeled target cells. After incubation for4hat37°C under 5% ϫ ␮ human NK cell activation (35). CO2 in air, the cells were pelleted at 250 g for 5 min, 100 lofthe In this paper, we investigated whether the AP-1 pathway is ac- supernatants was removed, and their radioactivity was measured. The per- centage of specific lysis was calculated by the following equation: (a Ϫ tivated in human NK cells upon 2B4 stimulation. AP-1 is an im- b/c Ϫ b) ϫ 100, where a is the radioactivity of the supernatant of target portant regulator of nuclear gene expression in leukocytes, having cells mixed with effector cells, b is that in the supernatant of target cells been found responsive to a wide range of stimuli and regulating a incubated alone, and c is that in the supernatant after lysis of target cells large number of genes (36, 37).
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