- Email: [email protected]
Stimulation of human mast cells by activated T cells leads to N-Ras activation through Ras guanine nucleotide releasing protein 1
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this may be explained, as recently shown, by the finding that lymphocytes carrying the PTPN22 variant had decreased responses to stimulation by anti-CD3 and by anti-IgM as well as decreased numbers of memory B cells (CD191CD271 population).9 In the future, similar additional abnormalities may eventually evolve in patients with CMCC with the PTPN22 variant 1858T. None of the patients had lymphopenia, and lymphocyte markers showed normal numbers of CD3, CD4, CD8, CD19, and CD56-positive cells in 4 patients. One patient had a low CD4 count (P2), and another had a relatively low number of B cells (P6). In vitro mitogenic responses to phytohemagglutinin or antiCD3 antibody were comparable to controls, but proliferative responses to Candida were low or absent in 4 of 6 patients. In contrast, only 1 of 8 patients with the common allele showed low responses to tetanus vaccine, and another patient had mild lymphopenia with a count of 1040 cells/mL. As expected, the APECED group of patients exhibited only an isolated defect in cellular response to Candida antigen (Table III). In summary, we have identified a group of patients who carry the PTPN22 variant allele 1858T, who in addition to chronic candidiasis frequently have recurrent microbial infections caused by antibody deficiency as well as gonadal failure and hypothyroidism. Amit Nahum, MD, PhD Andrea Bates, BSc Nigel Sharfe, PhD Chaim M. Roifman, MD From the Canadian Centre for Primary Immunodeficiency and the Division of Immunology and Allergy, Department of Pediatrics, The Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada. E-mail: [email protected]. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.
REFERENCES 1. Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, et al. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 2004;36:337-8. 2. Cohen S, Dadi H, Shaoul E, Sharfe N, Roifman CM. Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase. Lyp. Blood 1999;93:2013-24. 3. Cloutier JF, Veillette A. Cooperative inhibition of T-cell antigen receptor signaling by a complex between a kinase and a phosphatase. J Exp Med 1999;189: 111-21. 4. Gjo¨rloff-Wingren A, Saxena M, Williams S, Hammi D, Mustelin T. Characterization of TCR-induced receptor-proximal signaling events negatively regulated by the protein tyrosine phosphatase PEP. Eur J Immunol 1999;29:3845-54. 5. Begovich AB, Carlton VE, Honigberg LA, Schrodi SJ, Chokkalingam AP, Alexander HC, et al. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004;75:330-7. 6. Wu H, Cantor RM, Graham DS, Lingren CM, Farwell L, Jager PL, et al. Association analysis of the R620W polymorphism of protein tyrosine phosphatase PTPN22 in systemic lupus erythematosus families: increased T allele frequency in systemic lupus erythematosus patients with autoimmune thyroid disease. Arthritis Rheum 2005; 52:2396-402. 7. Ahonen P, Mylla¨rniemi S, Sipila I, Perheentupa J. Clinical variation of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990;322:1829-36. 8. Chapman SJ, Khor CC, Vannberg FO, Maskell NA, Davies CW, Hedley EL, et al. PTPN22 and invasive bacterial disease. Nat Genet 2006;38:499-500. 9. Rieck M, Arechiga A, Onengut-Gumuscu S, Greenbaum C, Concannon P, Buckner JH. Genetic variation in PTPN22 corresponds to altered function of T and B lymphocytes. J Immunol 2007;179:4704-10. doi:10.1016/j.jaci.2008.10.027
Stimulation of human mast cells by activated T cells leads to N-Ras activation through Ras guanine nucleotide releasing protein 1 To the Editor: We have recently identified and characterized a novel mast cell (MC) activation pathway initiated by contact with activated T cells.1 It has been reported that this pattern of activation is associated with phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase and p38.2 We showed that the cytokines IL-8 and oncostatin M are released from MCs after contact with activated T cells and that this process is subject to MAPK inhibition.3,4 The best characterized upstream regulator of the MAPK system is the small guanosine triphosphate (GTP)–binding protein Ras.5 This protein has critical functions in many cell types; however, in MCs its functions are not entirely understood. Therefore, and on the basis of our previous findings, we decided to study the spatiotemporal pattern of Ras activation in MCs stimulated by activated T cells. Ras is a family of small GTP-binding proteins composed of 3 closely related isoforms: H-Ras, N-Ras, and K-Ras.5 These isoforms are expressed in most mammalian cells yet exhibit different biologic outputs: K-Ras mutations are frequently seen in tumors, N-Ras is the central isoform in lymphocytes, and H-Ras regulates angiogenesis.5 First, we analyzed the expression levels of the 3 Ras isoforms in different human MC lines. We used LAD2 and HMC-1 MC lines and Jurkat T cells as the control.3,6,7 The endogenous levels of the different isoforms were analyzed by means of immunoblotting with specific antibodies. As shown in Fig 1, A, and similar to what has been reported in T cells, both MC lines expressed N-Ras and K-Ras but not H-Ras. The specificity of the antibodies was tested against overexpressed Ras isoforms in LAD2 cells (Fig 1, B). Ras is a molecular switch and as such can bind either GTP or guanosine diphosphate (GDP). The GTP-binding state activates Ras, whereas the replacement by the nucleotide GDP deactivates it.5 We therefore further analyzed whether Ras was activated after contact with T cells. LAD2 cells were incubated with membranes derived from resting or activated (50 ng/mL phorbol 12-myrtistate 13-acetate [PMA] for 60 minutes followed by extensive washing) T cells or by FCeRI cross-linking.4,8 Activated Ras molecules were affinity precipitated with the GST–Ras-binding domain (RBD) pull-down assay.7 Total Ras was immunoprecipitated with pan-Ras antibody. As shown in Fig 1, C, the total levels of Ras were similar in all conditions. However, differences were noticed in the fractions of GTP-loaded Ras. In both resting MCs and MCs incubated with membranes from resting T cells, the pool of activated Ras was faintly detected. When MCs were stimulated by FCeRI cross-linking, Ras was found to be activated. Likewise, incubation of MCs with activated T-cell membranes resulted in Ras activation (Fig 1, C). To further support these findings and to study the localization of activated Ras, we used imaging techniques. LAD2 cells were transiently transfected with cyan fluorescent protein (CFP)– tagged N-Ras and the florescent probe for activated Ras, yellow fluorescent protein (YFP)–labeled RBD.7 After 48 hours, the cells were treated as indicated in Fig 1, D, and imaged alive with confocal microscopy. As shown in Fig 1, D, we were able to detect mild recruitment of the probe to the Golgi apparatus in all
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FIG 1. Ras is activated in MCs after contact with T cells. The expression level of the 3 Ras isoforms was tested in the MC lines LAD2 and HMC-1 and the Jurkat T-cell line. A, The Ras isoforms levels were analyzed by means of immunoblotting with isoform-specific antibodies. B, The specificity of the antibodies was tested against overexpressed Ras isoforms in LAD2 cells. LAD2 cells were simulated as indicated for 5 minutes, and GTP-loaded Ras was affinity precipitated with the GST-RBD pull-down assay. Total Ras was immunoprecipitated with pan-Ras antibody. C, Both precipitates were analyzed by means of Western blotting with pan-Ras antibody. LAD2 cells were transfected with CFP-N-Ras and YFP-RBD. After 48 hours, the cells were stimulated as indicated and imaged alive. D, In all the conditions, CFP-N-Ras localized to the Golgi apparatus and to the PM. E, Resting Jurkat cells were transfected with YFP-RBD and CFP-GalT and imaged alive. F, PMA-activated Jurkat T cells (labeled with CMTPX-Red) were cocultured with LAD2 cells expressing N-Ras and YFP-RBD and imaged alive after 15 minutes.
conditions, suggesting constitutively active Ras at that compartment. To verify that YFP-RBD was localized to the Golgi apparatus, we transfected LAD2 cells with a specific Golgi marker, CFP-galactosyltransferase. As shown in Fig 1, E, YFP-RBD clearly decorated the Golgi apparatus. In 20% of the cells stimulated by FCeRI, we detected recruitment of activated Ras to the plasma membrane (PM; Fig 1, D), whereas the remaining cells were unchanged. However, when the cells were stimulated with membranes from activated T cells, the majority of the cells (70%) demonstrated activation of Ras at the PM (Fig 1, D). To
show that the effect is specific to T cells and to exclude residual PMA activity, we treated the cells with membranes derived from PMA-activated HeLa fibroblasts. As shown in Fig 1, D, there was no effect on Ras activation. Finally, to show that Ras activation can also be mediated by whole T cells (and not only by cell membranes), we cocultured LAD2 cells with PMA-stimulated Jurkat T cells. As shown in Fig 1, F, contact between LAD2 cells and activated Jurkat T cells resulted in N-Ras activation. Thus N-Ras is activated at the PM of MCs as a result of contact with activated T cells.
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FIG 2. RasGRP1 is recruited to the PM of MCs after contact with activated T cells. LAD2 cells were transfected with YFP-RasGRP1 or YFP-RasGRP4. A, Forty-eight hours later, the cells were treated as indicated for 5 minutes and imaged alive. B, LAD2 cells were transfected with YFP-RasGRP1 and cocultured with PMA-stimulated Jurkat T cells (labeled with CMTPX-Red). The cells were imaged after 15 minutes. C, Short inhibitory RNAs were used to knock down RasGRP1 in cells overexpressing N-Ras and YFP-RBD. Short inhibitory RNAs against a scramble sequence were used as a control. Forty-eight hours later, the cells were stimulated as indicated for 5 minutes and imaged alive.
The exchange of GTP/GDP nucleotides is assisted by a set of proteins called guanine exchange factors (GEFs).9,10 We therefore decided to study which of the 2 commonly investigated GEFs in MCs is accountable for Ras activation under these conditions.11,12 To answer this question, we overexpressed the GEFs Ras guanine nucleotide releasing proteins (RasGRPs) 1 or 4 tagged with YFP in LAD2 cells.9 Forty-eight hours later, the cells were treated as indicated in Fig 2 and imaged alive. As can be seen in Fig 2, A, RasGRP1 in resting cells distributed homogeneously throughout the cytosol and was excluded from the nucleus. In 95% of the cells treated with PMA, RasGRP1 translocated to the PM, thus demonstrating the ability of the protein to translocate to that compartment. In 80% of the cells stimulated through FCeRI, RasGRP1 distribution was unaffected, whereas in 20% of the cells, the protein translocated to the PM (Fig 2, A, insert). When the cells were treated with membranes from resting T cells, the RasGRP1 distribution pattern was unchanged. Interestingly, when the cells were treated with membranes obtained from activated T cells, we observed translocation to the PM in 85% of the cells. As for RasGRP4, we were unable to detect its translocation after stimulation with either FCeRI or with activated T cells. To document that this effect is not specific to T-cell membranes, we incubated LAD2 cells, expressing YFP-RasGRP1, with whole Jurkat T cells stimulated with PMA. As shown in Fig 2, B, RasGRP1 translocated to the PM in LAD2 cells after contact with activated T cells. The same pattern of activation was observed when LAD2 cells were cocultured with antiCD3–stimulated Jurkat T cells (data not shown). To confirm the necessity of RasGRP1 in this pathway, we used short inhibitory RNAs to knock down RasGRP1.7 We transfected
LAD2 cells with N-Ras, YFP-RBD, and short inhibitory RNAs targeting either scramble or RasGRP1 sequences. Forty-eight hours later, the cells were stimulated as indicated and imaged alive. As shown in Fig 2, C, the scramble sequence did not interfere with the activation of Ras at the PM, whereas knocking down RasGRP1 blocked the activation completely. It can be concluded that RasGRP1 and not RasGRP4 is accountable for Ras activation at the PM of MCs on contact with activated T cells. Ras proteins have critical functions in many cell types.5 However, in MCs their functions are not completely understood. In this study we show that Ras participates in the non-FCeRI activation pathway (ie, after contact with activated T cells). The RasGRPs are closely related GEFs, and in different cell types they have different functions. RasGRP1 is essential for T-cell receptor signaling, RasGRP2 is important for normal platelet function, and RasGRP3 is key to B-cell selection.9,10 Which of the RasGRPs is more important in MCs is unknown. Splice variants of RasGRP4 were reported in cells isolated from patients with mastocytosis and asthma.11 In addition, a study of leukemic MCs revealed nonfunctional RasGRP4, suggesting a role for this protein in MC homeostasis.11 Nevertheless, other investigators suggest that RasGRP1 is the important GEF in MCs. Liu et al12 showed an essential role for RasGRP1 in IgE-mediated allergic response. Degranulation, microtubule formation, and cytokine production in MCs from RasGRP1-deficient mice were disrupted.12 It is likely that different GEFs have different functions in MCs; however, our results support a role for RasGRP1, but not RasGRP4, downstream contact with activated T cells. To the best of our knowledge, this is the first documentation of the spatiotemporal pattern of Ras activation in live MCs. In
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addition, our data reveal that N-Ras and K-Ras, but not H-Ras, are the dominant isoforms in human MCs. We have previously shown that T cells could activate MCs by means of heterotypic adhesion.1-4 This pattern of activation involves the MAPK5 system and resulted in release of different cytokines. In this study we report that N-Ras is activated downstream of this pathway and is localized to the PM. The question as to which of the 2 GEFs, RasGRP1 or RasGRP4, is principal in MCs is still a matter of debate. Our data support a crucial role for RasGRP1. This work suggests that targeting the Ras pathway might be a possible treatment option for conditions in which MCs interact with T cells, such as sarcoidosis, rheumatoid arthritis, and graft tolerance. We thank Dr Mark Philips (New York University School of Medicine) for helpful consultation, discussions, and reagents. Irit Shefler, PhDa Yoseph A. Mekori, MDa,b,c Adam Mor, MDa,b From athe Laboratory of Allergy and Clinical Immunology and bthe Department of Medicine, Meir General Hospital, Kfar Saba, Israel, and cthe Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. E-mail: [email protected]. Supported by the Morasha program of the Israel Science Foundation (grant no. 1822/07). Adam Mor has received grant support form the Israel Science Foundation. Yoseph Mekori has received grant support from the Israel Science Foundation and Tel Aviv University and is incumbent of the F. Reiss Chair in Dermatology, Tel Aviv University. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.
REFERENCES 1. Inamura N, Mekori YA, Bhattacaryya SP, Bianchine PJ, Metcalfe DD. Induction and enhancement of FCeRI-dependent mast cell degranulation following coculture with activated T cells: dependency on ICAM-1 and leukocyte function-associated antigen (LFA)-1-mediated heterotypic aggregation. J Immunol 1998;160:4026-33. 2. Brill A, Baram D, Sela U, Salamon P, Mekori YA, Hershkovis R. Induction of mast cell interactions with blood vessel wall components by direct contact with intact T cells or T cell membranes in vitro. Clin Exp Allergy 2004;34:1725-31. 3. Salamon P, Shoham NG, Gavrieli R, Wolach B, Mekori YA. Human mast cells release Interleukin-8 and induce neutrophil chemotaxis on contact with activated T cells. Allergy 2005;60:1316-9. 4. Salamon P, Shoham NG, Puxeddu I, Paitan Y, Levi-schaffer F, Mekori YA. Human mast cells release oncostatin M on contact with activated T cells: possible biologic relevance. J Allergy Clin Immunol 2008;121:448-55. 5. Mor A, Philips MR. Compartmentalized Ras/MAPK signaling. Annu Rev Immunol 2006;24:771-800. 6. Kirshenbaun AS, Akin C, Wu Y, Rottem M, Goff JP, Beaven MA, et al. Characterization of novel stem cell factor responsive human mast cell lines LAD1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FCeRI or FCgRI. Leuk Res 2002;27:677-82. 7. Mor A, Campi G, Du G, Zheng Y, Foster DA, Dustin ML, et al. The lymphocyte function-associated antigen-1 receptor costimulates PM Ras via phospholipase D2. Nat Cell Biol 2007;9:713-9. 8. Baram D, Vaday GG, Salamon P, Drucker I, Hershkovis R, Mekori YA. Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-a. J Immunol 2001;167:4008-16. 9. Bivona TG, Perez De Castro I, Ahearn IM, Grana TM, Chiu VK, Lockyet PJ, et al. Phospholipase C gamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature 2003;424:624-5. 10. Stone JC. Regulation of Ras in lymphocytes: get a GRP. Biochem Soc Trans 2006; 34:858-61. 11. Yang Y, Li L, Wong GW, Krilis SA, Madhusudhan MS, Sali A, et al. RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium and diacylglycerol binding motifs. J Biol Chem 2002;277:25756-74. 12. Liu Y, Shu M, Nishida K, Hirano T, Zhang W. An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response. J Exp Med 2007;204:93-103. Available online August 29, 2008. doi:10.1016/j.jaci.2008.07.024
ORMDL3 variants associated with asthma susceptibility in North Americans of European ancestry To the Editor: Asthma is the most common chronic disease in children across all developed countries. Although the cause of the disease remains unknown, it is recognized as a complex genetic disorder with an environmental component.1,2 As with many other complex diseases, a long list of genes has been associated with asthma through linkage and candidate gene association studies, the majority of which do not replicate.1 The first genome-wide association study of asthma predisposition was recently published.3 In that study 317,000 single nucleotide polymorphisms (SNPs) were typed in 994 patients with childhood-onset asthma, resulting in the identification of a novel locus on chromosome 17q12-q21 containing multiple genes and associated markers. Expression analysis in lymphoblastoid cell lines revealed that ORMDL3 expression was strongly correlated with the asthma-associated variants, leading the authors to conclude that it was the most likely candidate gene at this locus. ORMDL3 encodes a 4-transmembrane domain–containing protein that is localized to the endoplasmic reticulum membrane.4 Although current knowledge of ORMDL3 function is limited, recent studies in yeast suggest the gene product might be involved in protein folding. To determine whether ORMDL3 is a genetic risk factor for the development of asthma in North American white subjects, we sought to replicate the association with the 10 most significantly associated SNPs in the study by Moffatt et al3 in 2 large pediatric asthma cohorts, one comprising patients of Northern European decent and another comprising African American patients. Both cohorts were collected at the Children’s Hospital of Philadelphia (CHOP). This study was approved by the Institutional Review Board at CHOP. Parental informed consent was obtained from all participants in this study for the purpose of DNA collection and genotyping. All patients and control subjects reported in this study were recruited at the CHOP between 2006 and 2008. All subjects were resident in the Greater Philadelphia area. The study of white subjects included 807 patients with physician-diagnosed asthma and 2583 disease-free control subjects without asthma. The study of African American subjects included 1456 patients with physician-diagnosed asthma and 1973 control subjects without asthma. Both white and African American patients were given diagnoses by CHOP physicians in accordance with the American Thoracic Society criteria5 and had been prescribed medication to control their asthma. All control samples, both white and African American subjects, had no history of asthma or reactive airway disease and had never been prescribed asthma medications. In addition to self-reported ancestry status, all patients and control subjects were screened at ancestry informative markers using Markov Chain Monte Carlo algorithm, as implemented in STRUCTURE,6 to reduce the risk of population stratification. Genomic inflation of 1.08 in the white study and 1.1 for the African American study reflected minor background stratification. Mean age of the case cohort was as follows: white subjects, 8.6 years (s 5.8; 62% male and 38% female); African American subjects, 7.5 years (s 5.7; 57% male and 43% female). All control subjects were recruited by
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this may be explained, as recently shown, by the finding that lymphocytes carrying the PTPN22 variant had decreased responses to stimulation by anti-CD3 and by anti-IgM as well as decreased numbers of memory B cells (CD191CD271 population).9 In the future, similar additional abnormalities may eventually evolve in patients with CMCC with the PTPN22 variant 1858T. None of the patients had lymphopenia, and lymphocyte markers showed normal numbers of CD3, CD4, CD8, CD19, and CD56-positive cells in 4 patients. One patient had a low CD4 count (P2), and another had a relatively low number of B cells (P6). In vitro mitogenic responses to phytohemagglutinin or antiCD3 antibody were comparable to controls, but proliferative responses to Candida were low or absent in 4 of 6 patients. In contrast, only 1 of 8 patients with the common allele showed low responses to tetanus vaccine, and another patient had mild lymphopenia with a count of 1040 cells/mL. As expected, the APECED group of patients exhibited only an isolated defect in cellular response to Candida antigen (Table III). In summary, we have identified a group of patients who carry the PTPN22 variant allele 1858T, who in addition to chronic candidiasis frequently have recurrent microbial infections caused by antibody deficiency as well as gonadal failure and hypothyroidism. Amit Nahum, MD, PhD Andrea Bates, BSc Nigel Sharfe, PhD Chaim M. Roifman, MD From the Canadian Centre for Primary Immunodeficiency and the Division of Immunology and Allergy, Department of Pediatrics, The Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada. E-mail: [email protected]. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.
REFERENCES 1. Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, et al. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 2004;36:337-8. 2. Cohen S, Dadi H, Shaoul E, Sharfe N, Roifman CM. Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase. Lyp. Blood 1999;93:2013-24. 3. Cloutier JF, Veillette A. Cooperative inhibition of T-cell antigen receptor signaling by a complex between a kinase and a phosphatase. J Exp Med 1999;189: 111-21. 4. Gjo¨rloff-Wingren A, Saxena M, Williams S, Hammi D, Mustelin T. Characterization of TCR-induced receptor-proximal signaling events negatively regulated by the protein tyrosine phosphatase PEP. Eur J Immunol 1999;29:3845-54. 5. Begovich AB, Carlton VE, Honigberg LA, Schrodi SJ, Chokkalingam AP, Alexander HC, et al. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004;75:330-7. 6. Wu H, Cantor RM, Graham DS, Lingren CM, Farwell L, Jager PL, et al. Association analysis of the R620W polymorphism of protein tyrosine phosphatase PTPN22 in systemic lupus erythematosus families: increased T allele frequency in systemic lupus erythematosus patients with autoimmune thyroid disease. Arthritis Rheum 2005; 52:2396-402. 7. Ahonen P, Mylla¨rniemi S, Sipila I, Perheentupa J. Clinical variation of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990;322:1829-36. 8. Chapman SJ, Khor CC, Vannberg FO, Maskell NA, Davies CW, Hedley EL, et al. PTPN22 and invasive bacterial disease. Nat Genet 2006;38:499-500. 9. Rieck M, Arechiga A, Onengut-Gumuscu S, Greenbaum C, Concannon P, Buckner JH. Genetic variation in PTPN22 corresponds to altered function of T and B lymphocytes. J Immunol 2007;179:4704-10. doi:10.1016/j.jaci.2008.10.027
Stimulation of human mast cells by activated T cells leads to N-Ras activation through Ras guanine nucleotide releasing protein 1 To the Editor: We have recently identified and characterized a novel mast cell (MC) activation pathway initiated by contact with activated T cells.1 It has been reported that this pattern of activation is associated with phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase and p38.2 We showed that the cytokines IL-8 and oncostatin M are released from MCs after contact with activated T cells and that this process is subject to MAPK inhibition.3,4 The best characterized upstream regulator of the MAPK system is the small guanosine triphosphate (GTP)–binding protein Ras.5 This protein has critical functions in many cell types; however, in MCs its functions are not entirely understood. Therefore, and on the basis of our previous findings, we decided to study the spatiotemporal pattern of Ras activation in MCs stimulated by activated T cells. Ras is a family of small GTP-binding proteins composed of 3 closely related isoforms: H-Ras, N-Ras, and K-Ras.5 These isoforms are expressed in most mammalian cells yet exhibit different biologic outputs: K-Ras mutations are frequently seen in tumors, N-Ras is the central isoform in lymphocytes, and H-Ras regulates angiogenesis.5 First, we analyzed the expression levels of the 3 Ras isoforms in different human MC lines. We used LAD2 and HMC-1 MC lines and Jurkat T cells as the control.3,6,7 The endogenous levels of the different isoforms were analyzed by means of immunoblotting with specific antibodies. As shown in Fig 1, A, and similar to what has been reported in T cells, both MC lines expressed N-Ras and K-Ras but not H-Ras. The specificity of the antibodies was tested against overexpressed Ras isoforms in LAD2 cells (Fig 1, B). Ras is a molecular switch and as such can bind either GTP or guanosine diphosphate (GDP). The GTP-binding state activates Ras, whereas the replacement by the nucleotide GDP deactivates it.5 We therefore further analyzed whether Ras was activated after contact with T cells. LAD2 cells were incubated with membranes derived from resting or activated (50 ng/mL phorbol 12-myrtistate 13-acetate [PMA] for 60 minutes followed by extensive washing) T cells or by FCeRI cross-linking.4,8 Activated Ras molecules were affinity precipitated with the GST–Ras-binding domain (RBD) pull-down assay.7 Total Ras was immunoprecipitated with pan-Ras antibody. As shown in Fig 1, C, the total levels of Ras were similar in all conditions. However, differences were noticed in the fractions of GTP-loaded Ras. In both resting MCs and MCs incubated with membranes from resting T cells, the pool of activated Ras was faintly detected. When MCs were stimulated by FCeRI cross-linking, Ras was found to be activated. Likewise, incubation of MCs with activated T-cell membranes resulted in Ras activation (Fig 1, C). To further support these findings and to study the localization of activated Ras, we used imaging techniques. LAD2 cells were transiently transfected with cyan fluorescent protein (CFP)– tagged N-Ras and the florescent probe for activated Ras, yellow fluorescent protein (YFP)–labeled RBD.7 After 48 hours, the cells were treated as indicated in Fig 1, D, and imaged alive with confocal microscopy. As shown in Fig 1, D, we were able to detect mild recruitment of the probe to the Golgi apparatus in all
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FIG 1. Ras is activated in MCs after contact with T cells. The expression level of the 3 Ras isoforms was tested in the MC lines LAD2 and HMC-1 and the Jurkat T-cell line. A, The Ras isoforms levels were analyzed by means of immunoblotting with isoform-specific antibodies. B, The specificity of the antibodies was tested against overexpressed Ras isoforms in LAD2 cells. LAD2 cells were simulated as indicated for 5 minutes, and GTP-loaded Ras was affinity precipitated with the GST-RBD pull-down assay. Total Ras was immunoprecipitated with pan-Ras antibody. C, Both precipitates were analyzed by means of Western blotting with pan-Ras antibody. LAD2 cells were transfected with CFP-N-Ras and YFP-RBD. After 48 hours, the cells were stimulated as indicated and imaged alive. D, In all the conditions, CFP-N-Ras localized to the Golgi apparatus and to the PM. E, Resting Jurkat cells were transfected with YFP-RBD and CFP-GalT and imaged alive. F, PMA-activated Jurkat T cells (labeled with CMTPX-Red) were cocultured with LAD2 cells expressing N-Ras and YFP-RBD and imaged alive after 15 minutes.
conditions, suggesting constitutively active Ras at that compartment. To verify that YFP-RBD was localized to the Golgi apparatus, we transfected LAD2 cells with a specific Golgi marker, CFP-galactosyltransferase. As shown in Fig 1, E, YFP-RBD clearly decorated the Golgi apparatus. In 20% of the cells stimulated by FCeRI, we detected recruitment of activated Ras to the plasma membrane (PM; Fig 1, D), whereas the remaining cells were unchanged. However, when the cells were stimulated with membranes from activated T cells, the majority of the cells (70%) demonstrated activation of Ras at the PM (Fig 1, D). To
show that the effect is specific to T cells and to exclude residual PMA activity, we treated the cells with membranes derived from PMA-activated HeLa fibroblasts. As shown in Fig 1, D, there was no effect on Ras activation. Finally, to show that Ras activation can also be mediated by whole T cells (and not only by cell membranes), we cocultured LAD2 cells with PMA-stimulated Jurkat T cells. As shown in Fig 1, F, contact between LAD2 cells and activated Jurkat T cells resulted in N-Ras activation. Thus N-Ras is activated at the PM of MCs as a result of contact with activated T cells.
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FIG 2. RasGRP1 is recruited to the PM of MCs after contact with activated T cells. LAD2 cells were transfected with YFP-RasGRP1 or YFP-RasGRP4. A, Forty-eight hours later, the cells were treated as indicated for 5 minutes and imaged alive. B, LAD2 cells were transfected with YFP-RasGRP1 and cocultured with PMA-stimulated Jurkat T cells (labeled with CMTPX-Red). The cells were imaged after 15 minutes. C, Short inhibitory RNAs were used to knock down RasGRP1 in cells overexpressing N-Ras and YFP-RBD. Short inhibitory RNAs against a scramble sequence were used as a control. Forty-eight hours later, the cells were stimulated as indicated for 5 minutes and imaged alive.
The exchange of GTP/GDP nucleotides is assisted by a set of proteins called guanine exchange factors (GEFs).9,10 We therefore decided to study which of the 2 commonly investigated GEFs in MCs is accountable for Ras activation under these conditions.11,12 To answer this question, we overexpressed the GEFs Ras guanine nucleotide releasing proteins (RasGRPs) 1 or 4 tagged with YFP in LAD2 cells.9 Forty-eight hours later, the cells were treated as indicated in Fig 2 and imaged alive. As can be seen in Fig 2, A, RasGRP1 in resting cells distributed homogeneously throughout the cytosol and was excluded from the nucleus. In 95% of the cells treated with PMA, RasGRP1 translocated to the PM, thus demonstrating the ability of the protein to translocate to that compartment. In 80% of the cells stimulated through FCeRI, RasGRP1 distribution was unaffected, whereas in 20% of the cells, the protein translocated to the PM (Fig 2, A, insert). When the cells were treated with membranes from resting T cells, the RasGRP1 distribution pattern was unchanged. Interestingly, when the cells were treated with membranes obtained from activated T cells, we observed translocation to the PM in 85% of the cells. As for RasGRP4, we were unable to detect its translocation after stimulation with either FCeRI or with activated T cells. To document that this effect is not specific to T-cell membranes, we incubated LAD2 cells, expressing YFP-RasGRP1, with whole Jurkat T cells stimulated with PMA. As shown in Fig 2, B, RasGRP1 translocated to the PM in LAD2 cells after contact with activated T cells. The same pattern of activation was observed when LAD2 cells were cocultured with antiCD3–stimulated Jurkat T cells (data not shown). To confirm the necessity of RasGRP1 in this pathway, we used short inhibitory RNAs to knock down RasGRP1.7 We transfected
LAD2 cells with N-Ras, YFP-RBD, and short inhibitory RNAs targeting either scramble or RasGRP1 sequences. Forty-eight hours later, the cells were stimulated as indicated and imaged alive. As shown in Fig 2, C, the scramble sequence did not interfere with the activation of Ras at the PM, whereas knocking down RasGRP1 blocked the activation completely. It can be concluded that RasGRP1 and not RasGRP4 is accountable for Ras activation at the PM of MCs on contact with activated T cells. Ras proteins have critical functions in many cell types.5 However, in MCs their functions are not completely understood. In this study we show that Ras participates in the non-FCeRI activation pathway (ie, after contact with activated T cells). The RasGRPs are closely related GEFs, and in different cell types they have different functions. RasGRP1 is essential for T-cell receptor signaling, RasGRP2 is important for normal platelet function, and RasGRP3 is key to B-cell selection.9,10 Which of the RasGRPs is more important in MCs is unknown. Splice variants of RasGRP4 were reported in cells isolated from patients with mastocytosis and asthma.11 In addition, a study of leukemic MCs revealed nonfunctional RasGRP4, suggesting a role for this protein in MC homeostasis.11 Nevertheless, other investigators suggest that RasGRP1 is the important GEF in MCs. Liu et al12 showed an essential role for RasGRP1 in IgE-mediated allergic response. Degranulation, microtubule formation, and cytokine production in MCs from RasGRP1-deficient mice were disrupted.12 It is likely that different GEFs have different functions in MCs; however, our results support a role for RasGRP1, but not RasGRP4, downstream contact with activated T cells. To the best of our knowledge, this is the first documentation of the spatiotemporal pattern of Ras activation in live MCs. In
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addition, our data reveal that N-Ras and K-Ras, but not H-Ras, are the dominant isoforms in human MCs. We have previously shown that T cells could activate MCs by means of heterotypic adhesion.1-4 This pattern of activation involves the MAPK5 system and resulted in release of different cytokines. In this study we report that N-Ras is activated downstream of this pathway and is localized to the PM. The question as to which of the 2 GEFs, RasGRP1 or RasGRP4, is principal in MCs is still a matter of debate. Our data support a crucial role for RasGRP1. This work suggests that targeting the Ras pathway might be a possible treatment option for conditions in which MCs interact with T cells, such as sarcoidosis, rheumatoid arthritis, and graft tolerance. We thank Dr Mark Philips (New York University School of Medicine) for helpful consultation, discussions, and reagents. Irit Shefler, PhDa Yoseph A. Mekori, MDa,b,c Adam Mor, MDa,b From athe Laboratory of Allergy and Clinical Immunology and bthe Department of Medicine, Meir General Hospital, Kfar Saba, Israel, and cthe Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. E-mail: [email protected]. Supported by the Morasha program of the Israel Science Foundation (grant no. 1822/07). Adam Mor has received grant support form the Israel Science Foundation. Yoseph Mekori has received grant support from the Israel Science Foundation and Tel Aviv University and is incumbent of the F. Reiss Chair in Dermatology, Tel Aviv University. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.
REFERENCES 1. Inamura N, Mekori YA, Bhattacaryya SP, Bianchine PJ, Metcalfe DD. Induction and enhancement of FCeRI-dependent mast cell degranulation following coculture with activated T cells: dependency on ICAM-1 and leukocyte function-associated antigen (LFA)-1-mediated heterotypic aggregation. J Immunol 1998;160:4026-33. 2. Brill A, Baram D, Sela U, Salamon P, Mekori YA, Hershkovis R. Induction of mast cell interactions with blood vessel wall components by direct contact with intact T cells or T cell membranes in vitro. Clin Exp Allergy 2004;34:1725-31. 3. Salamon P, Shoham NG, Gavrieli R, Wolach B, Mekori YA. Human mast cells release Interleukin-8 and induce neutrophil chemotaxis on contact with activated T cells. Allergy 2005;60:1316-9. 4. Salamon P, Shoham NG, Puxeddu I, Paitan Y, Levi-schaffer F, Mekori YA. Human mast cells release oncostatin M on contact with activated T cells: possible biologic relevance. J Allergy Clin Immunol 2008;121:448-55. 5. Mor A, Philips MR. Compartmentalized Ras/MAPK signaling. Annu Rev Immunol 2006;24:771-800. 6. Kirshenbaun AS, Akin C, Wu Y, Rottem M, Goff JP, Beaven MA, et al. Characterization of novel stem cell factor responsive human mast cell lines LAD1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FCeRI or FCgRI. Leuk Res 2002;27:677-82. 7. Mor A, Campi G, Du G, Zheng Y, Foster DA, Dustin ML, et al. The lymphocyte function-associated antigen-1 receptor costimulates PM Ras via phospholipase D2. Nat Cell Biol 2007;9:713-9. 8. Baram D, Vaday GG, Salamon P, Drucker I, Hershkovis R, Mekori YA. Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-a. J Immunol 2001;167:4008-16. 9. Bivona TG, Perez De Castro I, Ahearn IM, Grana TM, Chiu VK, Lockyet PJ, et al. Phospholipase C gamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature 2003;424:624-5. 10. Stone JC. Regulation of Ras in lymphocytes: get a GRP. Biochem Soc Trans 2006; 34:858-61. 11. Yang Y, Li L, Wong GW, Krilis SA, Madhusudhan MS, Sali A, et al. RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium and diacylglycerol binding motifs. J Biol Chem 2002;277:25756-74. 12. Liu Y, Shu M, Nishida K, Hirano T, Zhang W. An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response. J Exp Med 2007;204:93-103. Available online August 29, 2008. doi:10.1016/j.jaci.2008.07.024
ORMDL3 variants associated with asthma susceptibility in North Americans of European ancestry To the Editor: Asthma is the most common chronic disease in children across all developed countries. Although the cause of the disease remains unknown, it is recognized as a complex genetic disorder with an environmental component.1,2 As with many other complex diseases, a long list of genes has been associated with asthma through linkage and candidate gene association studies, the majority of which do not replicate.1 The first genome-wide association study of asthma predisposition was recently published.3 In that study 317,000 single nucleotide polymorphisms (SNPs) were typed in 994 patients with childhood-onset asthma, resulting in the identification of a novel locus on chromosome 17q12-q21 containing multiple genes and associated markers. Expression analysis in lymphoblastoid cell lines revealed that ORMDL3 expression was strongly correlated with the asthma-associated variants, leading the authors to conclude that it was the most likely candidate gene at this locus. ORMDL3 encodes a 4-transmembrane domain–containing protein that is localized to the endoplasmic reticulum membrane.4 Although current knowledge of ORMDL3 function is limited, recent studies in yeast suggest the gene product might be involved in protein folding. To determine whether ORMDL3 is a genetic risk factor for the development of asthma in North American white subjects, we sought to replicate the association with the 10 most significantly associated SNPs in the study by Moffatt et al3 in 2 large pediatric asthma cohorts, one comprising patients of Northern European decent and another comprising African American patients. Both cohorts were collected at the Children’s Hospital of Philadelphia (CHOP). This study was approved by the Institutional Review Board at CHOP. Parental informed consent was obtained from all participants in this study for the purpose of DNA collection and genotyping. All patients and control subjects reported in this study were recruited at the CHOP between 2006 and 2008. All subjects were resident in the Greater Philadelphia area. The study of white subjects included 807 patients with physician-diagnosed asthma and 2583 disease-free control subjects without asthma. The study of African American subjects included 1456 patients with physician-diagnosed asthma and 1973 control subjects without asthma. Both white and African American patients were given diagnoses by CHOP physicians in accordance with the American Thoracic Society criteria5 and had been prescribed medication to control their asthma. All control samples, both white and African American subjects, had no history of asthma or reactive airway disease and had never been prescribed asthma medications. In addition to self-reported ancestry status, all patients and control subjects were screened at ancestry informative markers using Markov Chain Monte Carlo algorithm, as implemented in STRUCTURE,6 to reduce the risk of population stratification. Genomic inflation of 1.08 in the white study and 1.1 for the African American study reflected minor background stratification. Mean age of the case cohort was as follows: white subjects, 8.6 years (s 5.8; 62% male and 38% female); African American subjects, 7.5 years (s 5.7; 57% male and 43% female). All control subjects were recruited by